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2 Published and copyright by: Medical Assotiation of Zenica-Doboj Canton; Address: Zenica, 72000, Bulevar kralja Tvrtka I 4, Bosnia and Herzegovina; tel./fax: ; ljkozedo@bih.net.ba, web site: http// For ordering information please contact: Tatjana Žilo, ljkozedo@bih.net.ba; Access to this journal is available free online trough: The Journal is indexed by MEDLINE, EMBASE (Exerpta Medica), Scopus, EBSCO; Directory of Research Journals Indexing (DRJI); ISSN DTP by: Graphic and web design studio B Panel Zenica, Zmaja od Bosne bb, info@bpanel.ba, tel , ; Printed by: GRIN d.o.o. Ulica Patriotske lige bb, Gračanica, Bosna i Hercegovina; Tel.: ; info@grin.ba; direktor@grin.ba; Printing supported by the Federal Ministry of Education and Science (Federalno ministarstvo obrazovanja i nauke, BiH)

3 Medicinski Glasnik Official Publication of the Medical Association of Zenica-Doboj Canton Bosnia and Herzegovina Editor-in-chief Selma Uzunović Zenica, Bosnia and Herzegovina MANAGING EDITOR Tarik Kapidžić Zenica, Bosnia and Herzegovina Editors Adem Balić, Tuzla, Bosnia and Herzegovina Dubravka Bartolek, Zagreb, Croatia Branka Bedenić, Zagreb, Croatia Asja Čelebić, Zagreb, Croatia Josip Čulig, Zagreb, Croatia Filip Čulo, Mostar, Bosnia and Herzegovina Jordan Dimanovski, Zagreb, Croatia Branko Dmitrović, Osijek, Croatia Ines Drenjančević, Osijek, Croatia Harun Drljević, Zenica, Bosnia and Herzegovina Davorin Đanić, Slavonski Brod, Croatia Lejla Ibrahimagić-Šeper, Zenica, Bosnia and Herzegovina Tatjana Ille, Belgrade, Serbia Vjekoslav Jerolimov, Zagreb, Croatia Mirko Šamija, Zagreb, Croatia Sven Kurbel, Osijek, Croatia Snježana Pejičić, Banja Luka, Bosnia and Herzegovina Belma Pojskić, Zenica, Bosnia and Herzegovina Besim Prnjavorac, Tešanj, Bosnia and Herzegovina Asja Prohić, Sarajevo, Bosnia and Herzegovina Velimir Profozić, Zagreb, Croatia Radivoje Radić, Osijek, Croatia Amira Redžić, Sarajevo, Bosnia and Herzegovina Suad Sivić, Zenica, Bosnia and Herzegovina Sonja Smole-Možina, Ljubljana, Slovenia Vladimir Šimunović, Mostar, Bosnia and Herzegovina Adrijana Vince, Zagreb, Croatia Jasmina Vraneš, Zagreb, Croatia Živojin Žagar, Zagreb, Croatia Secretary: Tatjana Žilo; Proofreaders: Aras Borić (Bosnian, Croatian, Serbian), Glorija Alić (English)

4 MEDICINSKI GLASNIK Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 12, Number 2, August 2016 Free full-text online at: and (DOAJ, Directory of Open Access Journals) Original article 108 Effects of post-sampling analysis time, type of blood samples and collection tubes on values of blood gas testing Jasmina Smajić, Damira Kadić, Sabaheta Hasić, Nafija Serdarević 113 Association of LPIN1 gene variations with markers of metabolic syndrome in population from Bosnia and Herzegovina Tamer Bego, Tanja Dujić, Barbara Mlinar, Sabina Semiz, Maja Malenica, Besim Prnjavorac, Barbara Ostanek, Janja Marc, Anida Čaušević-Ramoševac, Adlija Čaušević 122 Alpha-lipoic acid reduces body weight and regulates triglycerides in obese patients with diabetes mellitus Azra Okanović, Besim Prnjavorac, Edin Jusufović, Rifat Sejdinović 128 Positive correlation between uric acid and C-reactive protein serum level in healthy individuals and patients with acute coronary syndrome Emina Spahić1, Sabaheta Hasić2, Emina Kiseljaković2, Halima Resić3, Mehmed Kulić4 133 Role of echocardiography in diagnosis and management of complete papillary muscle rupture caused by myocardial infarction Josip Vincelj, Stanko Biočić, Mario Udovičić, Mario Sičaja, Sandra Jakšić Jurinjak 139 Impact of reperfusion therapy and infarct localization on frequency of premature ventricular beats in acute myocardial infarction Davor Horvat, Josip Vincelj 144 Possibilities of differentiation of solitary focal liver lesions by computed tomography perfusion Irmina Sefić Pašić, Anes Pašić, Spomenka Kristić, Adnan Beganović, Aladin Čarovac, Amra Džananović, Lidija Lincender, Sandra Vegar Zubović 151 Familial autoimmune thyroid disease and PTPN-22 Gabriel Conzuelo Rodríguez, Hugo Mendieta Zerón 157 Methicillin-resistant S. aureus (MRSA), extended-spectrum (ESBL)- and plasmid-mediated AmpC ß-lactamase -producing Gram-negative bacteria associated with skin and soft tissue infections in hospital and community settings Selma Uzunović, Branka Bedenić, Ana Budimir, Amir Ibrahimagić, Farah Kamberović, Zlatko Fiolić, Michelle I. A. Rijnders, Ellen E. Stobberingh 169 Emergence of extensive drug-resistant (XDR) Acinetobacter baumanniiin the Clinical Center University of Sarajevo, Bosnia and Herzegovina Amela Dedeić-Ljubović, Ðana Granov, Mirsada Hukić 177 Brucellosis in children in Bosnia and Herzegovina in the period Sead Ahmetagić, Humera Porobić-Jahić, Nada Koluder, Lejla Čalkić, Snježana Mehanić, Eldira Hadžić, Nevzeta Ibrahimpašić, Svjetlana Grgić, Mirela Zirić, Jelena Bajić, Denis Žepić 183 Differences in newborn umbilical cord care Sanja Kanisek, Nada Prlić, Ivana Barać, Lorna Dubac Nemet 190 Effects of perioperative statin treatment on postoperative atrial fibrillation and cardiac mortality in patients undergoing coronary artery bypass grafting: a propensity score analysis Ayşegül Kunt, Sedat Özcan, Aslihan Küçüker, Dolunay Odabaşi, Alper Sami Kunt 196 Efficacy of chronic statin therapy on major cardiac events after coronary artery bypass grafting: low-dose versus high-dose Ayşegül Kunt, Sedat Özcan, Aslihan Küçüker, Dolunay Odabaşi, Alper Sami Kunt

5 202 Incidence of endophthalmitis after intravitreal application of anti VEGF therapy at the University Clinical Center in Tuzla, Bosnia and Herzegovina Svjetlana Terzić, Adisa Pilavdžić, Amra Nadarević Vodenčarević 206 Therapeutic efficacy and toxicity of bolus application of chemotherapy protocol in the treatment of metastatic colorectal cancer Ibrahim Šišić, Belma Pojskić, Alma Mekić-Abazović, Vladimir Kovčin 212 Differences in body mass index and height factors between men with and without varicocele Hamid Shafi, Mouloud Agajani Delavar 216 Genetic polymorphisms variants in interleukin-6 and interleukin-1beta patients with obstructive sleep apnea syndrome in East Northern Turkey Ilhami Gok, Nergiz Huseyinoglu, Dogan Ilhan Medicinski Glasnik is indexed by MEDLINE, EMBASE (Exerpta Medica), EBSCO, Scopus, and Directory of Research Journals Indexing (DRJI)

6 ORIGINAL ARTICLE Effects of post-sampling analysis time, type of blood samples and collection tubes on values of blood gas testing Jasmina Smajić¹, Damira Kadić², Sabaheta Hasić³, Nafija Serdarević¹ ¹Clinical Chemistry and Biochemistry, Clinical Centre University of Sarajevo, Sarajevo, ²Department of Laboratory Diagnostics, Cantonal Hospital Zenica, Zenica, ³Department of Medical Biochemistry, School of Medicine, University of Sarajevo, Sarajevo; Bosnia and Herzegovina ABSTRACT Aim To investigate effects of post-sampling analysis time, a type of blood samples and collection tubes on blood gas testing. Corresponding author: Jasmina Smajić Clinical Chemistry and Biochemistry, Clinical Centre University of Sarajevo Bolnička 25, Sarajevo, Bosnia and Herzegovina Phone: ; Fax: ; jasmina.smajic@yahoo.com Original submission: 27 May 2015; Revised submission: 06 July 2015; Accepted: 08 July doi: / Methods This study included 100 patients at the Clinic for Pulmonary Diseases, Clinical Centre Sarajevo. The partial pressure of oxygen (po 2 ) and carbon dioxide (pco 2 ), and the oxygen saturation level of hemoglobin (so 2 ) were analyzed in the arterial blood samples (ABS) and capillary blood samples (CBS) by a potentiometric method using a blood gas analyzer ABL 555 (Radiometer, Copenhagen, Denmark). Paired measurements of ABS were performed within 15 minutes and after 60 minutes from sampling and compared. The results of CBS obtained within 15 minutes were compared with matching ABS results, as well as the results obtained from CBS within 15 minutes taken into glass and plastic tubes. Results po 2 and so 2 values were significantly lower after 60 minutes compared to those within 15 minutes in ABS (9.20±1.89 vs. 9.51±1.95 and 91.25±5.03 vs ±4.5; p<0.01, respectively). Values of po 2 and so 2 in CBS were significantly lower than values obtained in ABS (8.92±2.07 vs. 9.51±1.95 and 91.25±4.86 vs ±4.50; p<0.01, respectively). Obtained po 2 and so 2 values in CBS in the plastic tubes were higher than those in the glass tubes (8.50±1.98 vs. 7.89±2.0 and 89.66±11.04 vs ±11.22, p<0.01 respectively). pco 2 blood values were not influenced significantly (p>0.05). Conclusion The length of post-sampling analysis time, a type of blood samples and collection tubes have significant impact on blood oxygen parameters. Analysis within 15 minutes after blood sampling is considered as appropriate. Key words: blood gas analysis, capillary tubing, syringes, glass, plastics. Med Glas (Zenica) 2015; 12(2):

7 Smajić et al. The influences on blood gas testing INTRODUCTION Blood gas testing is a common analysis in critically ill patients that provides important information for their treatment. It comprises an analysis of partial pressures of oxygen (po 2 ) and carbon dioxide (pco 2 ), blood ph and oxygen saturation level of haemoglobin (so 2 ) in arterial, venous or capillary blood, depending on a clinical question. The interval between sampling and analysis, as well as a type of blood samples and collection tubes are important pre-analytical factors that can affect blood gas testing (1). The gold standard samples for blood gas analysis are arterial blood samples (ABS) collected from arterial catheter or by arterial puncture. Properly arterial blood sampling is difficult and painful for the patient, but a clinician is able to get a true picture about oxygen supply. Venous blood sampling is less painful and risky, but venous blood samples are not an adequate replacement for ABS in routine blood gas testing (2). Capillary blood samples (CBS), least painful and easiest to obtain, from the fingertip, heel or earlobe, can also be used in routine practice (3,4). Obtaining CBS is less invasive and can be performed by various healthcare professionals, while arterial blood sampling requires specially trained medical personnel (5). Proper time processing of blood samples is a critical pre-analytical step in the integrity of laboratory results because of continued metabolism in the blood that can alter blood gas values during the time between sampling and analysis or by diffusion of gases through the wall of the collection device (6,7). A type of collection tubes can also affect blood gas analysis (8). While glass tubes were common collection devices in the past, today plastic devices are mainly used because of safety concerns (1). The study aim was to investigate the effects of time interval between sampling and analysis, a type of blood samples and collection tubes on blood gas testing. PATIENTS AND METHODS The study included analysis of blood samples obtained from 100 hospitalized patients and outpatients at the Clinic for Pulmonary Diseases, Clinical Centre University of Sarajevo during a six-month period in From 50 patients, two types of blood samples, arterial and capillary, were collected at the same time. Arterial blood samples were collected by arterial puncture of radial artery into the heparinized plastic syringes, while CBS were collected by skin puncture of fingertip into heparinized capillary plastic tubes. From other 50 patients, CBS were collected both in glass and plastic heparinized capillary tubes at the same time. Samples were thoroughly mixed with metal stirrer and magnet to obtain proper anticoagulation, and to avoid contamination by clots. Care was taken that no air entered the collection devices. Arterial blood samples were analyzed within 15 minutes, and after 60 minutes from sampling. Capillary blood samples were analyzed within 15 minutes from sampling. Blood gas analyses of po 2, pco 2 and so 2 were performed at the room temperature. All determinations were performed at the Clinical Chemistry and Biochemistry, Clinical Centre University of Sarajevo by potentiometric method using a blood gas analyzer ABL 555 (Radiometer, Copenhagen, Denmark), according to the manufacturer s instructions. Tubes and syringes for blood sampling were purchased from Radiometer. The research was done respecting ethical standards of the Declaration of Helsinki. Ethics approval was obtained from the Ethics Committee of School of Medicine, University of Sarajevo. Variables are presented as a mean ± standard deviation. Comparison of means between two groups was analyzed by the Student t-test for independent samples. The paired-samples t-test has been used to compare the means between two related groups on the same continuous, dependent variable. Values of p<0.05 were considered as statistically significant. RESULTS One hundred arterial blood determinations from 50 patients were performed, e. g., 50 measurements for each of the two experimental conditions, within 15 minutes and after 60 minutes from sampling. Values of po 2 and so 2 in ABS after 60 minutes were significantly lower compared to those analyzed within 15 minutes (p=0.007 and p=0.0001, respectively), while no statistically si- 109

8 Medicinski Glasnik, Volume 12, Number 2, August 2015 gnificant differences were found for pco 2 values (p=0.17) (Table 1). Table 1. Arterial blood gas testing during two post-sampling time points Parameters 0-15 min after 60 min p po2 (kpa) 9.51± ± so2 (%) 92.40± ± pco2 (kpa) 5.36± ± po 2, partial pressure of oxygen; so 2, oxygen saturation level of hemoglobin; pco 2, partial pressure of carbon dioxide Blood gas testing in CBS within 15 minutes from blood sampling were performed for the same patients in order to investigate the influence of a sample type on blood gas values. Results showed significantly lower values of po 2 and so 2 in CBS compared to ABS (p=0.009 and p=0.0001, respectively), while no statistically significant differences were found for pco 2 values (p=0.348) (Table 2). Table 2. Arterial and capillary blood gas testing within 15 minutes of post-sampling period Parameters ABS CBS p po2 (kpa) 9.51 ± ± so2 (%) ± ± pco2 (kpa) 5.36 ± ± ABS, arterial blood samples; CBS, capillary blood samples; po 2, partial pressure of oxygen; so 2, oxygen saturation level of hemoglobin; pco 2, partial pressure of carbon dioxide For other 50 patients, blood gas analyses of CBS in glass and plastic tubes were performed, also within 15 minutes, in order to investigate the impact of a type of sampling tubes on blood gas values. Results showed significantly higher values of po 2 and so 2 in CBS in the plastic tubes compared to CBS in the glass tubes (p= and p=0.002, respectively), while no statistically significant differences were found for pco 2 values (p=0.278) (Table 3). Table 3. Capillary blood gas testing in different tube types Parameters Glass tube Plastic tube p po2 (kpa) 7.89 ± ± so2 (%) ± ± pco2 (kpa) 5.13 ± ± po 2, partial pressure of oxygen; so 2, oxygen saturation level of hemoglobin; pco 2, partial pressure of carbon dioxides. DISCUSSION The results of our study showed that arterial blood gas testing were affected by the length of postsampling time, but only po 2 and so 2 blood values showed statistically significant changes. Similar to our findings, previous studies described the influence of time between sampling and analysis on blood gas values. Beaulieu at al. concluded that po 2, in blood samples stored on ice, should be analyzed within 30 minutes compared to pco 2 which showed no clinically significant changes within 60 minutes (7). Knowles at al. found significantly higher po 2 values in the samples kept for 30 minutes compared to those analyzed immediately (8). Observed results can be explained by the oxygen diffusion through the wall of plastic syringes. Although insignificantly, Mohammadhoseini at al. showed that po 2 values in ABS stored for 60 minutes at 22 C were lower compared with those analyzed immediately. They also found statistically significant higher values of pco 2 in those samples but those changes were not clinically significant (9). Our study results are mainly consistent with the previous finding that post-sampling time significantly affects po 2 and so, so we have 2 to analyze these parameters immediately, or as soon as possible. Statistically significant decrease of po 2 and so 2 that we found in ABS analyzed after 60 minutes at room temperature indicates that metabolic consumption of oxygen exceeds its diffusion through the wall of plastic syringes. Values of po 2 and so 2 in CBS in our study were significantly lower in relation to those measured in ABS within 15 minutes. Statistically significant differences were not found for pco 2 values. Zavorski et al. showed that fingertip blood sample analysis can predict arterial pco 2, but not po 2 and so 2 (10). Murphy at al. found that agreement between measurements of arterial and capillary blood gases were good for pco 2, but poor for po 2 and that continuous pulse oximetry is more suitable for ensuring adequate and controlled oxygenation of the patient (11). Zavorsky et al. considered that CBS may be an appropriate replacement for ABS when analyzing po 2, except residual standard error of 6 mmhg is required for precision, and that capillary pco 2 accurately reflects arterial pco 2 over a wide range of values (12). Lower values of po 2 and so 2 in CBS are expected due to the fact that capillary blood is a mixture of capillary, arteriolar and venial blood as well as interstitial and intracellular fluid. Values of pco 2 have no significant differences in the ABS and CBS because of lower arteriovenous gradient (1). 110

9 Smajić et al. The influences on blood gas testing Comparison of our findings for so 2 and po 2 values in CBS within 15 minutes from sampling, between samples in the glass and plastic tubes showed that values obtained in the plastic tubes were higher compared to those in the glass tubes (p<0.01). Statistically significant differences were not found for pco 2 blood values. The plastic containers for blood gas testing are partially gas permeable with increasing oxygen permeability at lower temperature (7,8,13,14). Our findings confirmed that po₂ values in the plastic tubes at room temperature increase within 15 minutes, despite ongoing cellular metabolism which is dominant in the glass tube, because of the differences in gas partial pressures between blood and ambient. Historically implemented practice of keeping glass syringes on ice is not recommended for plastic syringes any more (1,9). Finally, we conclude that getting an accurate result for blood gas testing, special attention should be devoted to many pre-analytical activities. The length of post-sampling analysis interval has significant impact on blood oxygen parameters. Although there is no doubt that blood samples should be analyzed as soon as possible, analysis within 15 minutes of blood sampling is also con- sidered as appropriate. Even though the gold standard samples for blood gas testing are ABS, when the circumstances do not permit taking such samples, CBS can be taken as an alternative sample. On such occasions, po 2 and so 2 results should be taken with caution. For the patients requiring continuous monitoring of blood gases it is best to analyze ABS, but also it can be extremely beneficial to analyze CBS in combination with pulse oximetry. Since type of collection tubes also affect the oxygen parameters significantly, samples should be taken into glass tubes if we are not able to analyze it in appropriate time. ACKNOWLEDGEMENT The authors would like to thank Professor Hasan Žutić and all medical staff at the Clinic for Pulmonary Diseases and Clinical Chemistry and Biochemistry, Clinical Centre University of Sarajevo, who participated in the study. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATIONS Competing interests: none to declare. REFERENCES 1. Baird G. Preanalitical considerations in blood gas analysis. Biochem Med (Zagreb) 2013; 23: CLSI. Blood Gas and ph Analysis and Related Measurements; Approved Guideline - second edition. CLSI Document C46-A2. Wayne, PA: Clinical and Laboratory Standards Institute; Burnett RW, Covington AK, Fogh-Andersen N, Külpmann WR, Maas AH, Müller-Plathe O, Siggaard-Andersen O, Van Kessel AL, Wimberley PD, Zijlstra WG. International Federation of Clinical Chemistry (IFCC). Scientific Division. Committee on ph, Blood Gases and Electrolytes. Approved IFCC recommendations on whole blood sampling, transport and storage for simultaneous determination of ph, blood gases and electrolytes. Eur J Clin Chem Clin Biochem. 1995; 33: CLSI. Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard - sixth edition. CLSI Document H04- A6. Wayne, PA: Clinical Laboratory Standards Institute; Hardinge M, Annandale J, Bourne S, Cooper B, Evans A, Freeman D, Green A, Hippolyte S, Knowles V, MacNee W, McDonnell L, Pye K, Suntharalingam J, Vora V, Wilkinson T; British Thoracic Society Home Oxygen Guideline Development Group; British Thoracic Society Standards of Care Committee. British Thoracic Society guidelines for home oxygen use in adults. Thorax 2015; 70:i1-i Srisan P, Udomsri T, Jetanachai P, Lochindarat S, Kanjanapattanakul W. Effects of temperature and time delay on arterial blood gas and electrolyte measurements. J Med Assoc Thai 2011; 94: Beaulieu M, Lapointe Y, Vinet B. Stability of PO 2, PCO 2, and ph in fresh blood samples stored in plastic syringe with low heparin in relation to various blood-gas and hematological parameters. Clin Biochem 1999; 32: Knowles TP, Mullin RA, Hunter JA, Douce FH. Effects of syringe material, sample storage time, and temperature on blood gases and oxygen saturation in arterialized human blood samples. Respir Care 2006; 51: Mohammadhoseini E, Safavi E, Seifi S, Seifirad S, Firoozbakhsh S, Peiman S. Effect of Sample Storage Temperature and Time Delay on Blood Gases, Bicarbonate and ph in Human Arterial Blood Samples. Iran Red Crescent Med J 2015; 17: e Zavorsky GS, Lands LC, Schneider W, Carli F. Comparison of fingertip to arterial blood samples at rest and during exercise. Clin J Sport Med 2005; 15:

10 Medicinski Glasnik, Volume 12, Number 2, August Murphy R, Thethy S, Raby S, Beckley J, Terrace J, Fiddler C, Craig M, Robertson C. Capillary blood gases in acute exacerbations of COPD. Respir Med 2006; 100: Zavorsky GS, Cao J, Mayo NE, Gabbay R, Murias JM. Arterial versus capillary blood gases: a meta analysis. Respir Physiol Neurobiol 2007; 155: Smeenk FW, Janssen JD, Arends BJ, Harff GA, van den Bosch JA, Schönberger JP, Postmus PE. Effects of four different methods of sampling arterial blood and storage time on gas tensions and shunt calculation in the 100% oxygen test. Eur Respir J 1997; 10: Schmidt C, Müller-Plathe O. Stability of po2, pco2 and ph in heparinized whole blood samples: influence of storage temperature with regard to leukocyte count and syringe material. Eur J Clin Chem Clin Biochem 1992; 30: Učinak vremena proteklog od uzorkovanja do analize, vrste uzoraka krvi i cjevčica za uzorkovanje na analiziranje gasova u krvi Jasmina Smajić¹, Damira Kadić², Sabaheta Hasić³, Nafija Serdarević¹ ¹Klinička hemija i biohemija, Klinički centar Univerziteta u Sarajevu, Sarajevo, ²Služba za laboratorijsku dijagnostiku, Kantonalna bolnica Zenica, Zenica, ³Institut za medicinsku biohemiju, Medicinski fakultet Univerziteta u Sarajevu, Sarajevo; Bosna i Hercegovina SAŽETAK Cilj Istražiti učinke vremena od uzorkovanja krvi do analiziranja, vrste uzoraka krvi i cjevčica za uzorkovanje na analiziranje gasova u krvi. Metode Ispitivanje je uključivalo 100 pacijenata Klinike za plućne bolesti Kliničkog centra Univerziteta u Sarajevu. Parcijalni pritisak kiseonika (po 2 ) i ugljen dioksida (pco 2 ), te nivo saturacije hemoglobina kiseonikom (so 2 ) analizirani su u arterijskim i kapilarnim uzorcima krvi potenciometrijskom metodom na analizatoru ABL 555 (Radiometer, Kopenhagen, Danska). Upoređivana su uparena mjerenja uzoraka arterijske krvi izvođena unutar 15 minuta i nakon 60 minuta od uzorkovanja. Rezultati dobijeni iz kapilarnih uzoraka unutar 15 minuta upoređivani su s odgovarajućim rezultatima arterijskih uzoraka, kao i rezultati dobijeni iz kapilarnih uzoraka uzetih u roku 15 minuta u staklene i plastične cjevčice. Rezultati Vrijednosti po 2 i so 2 nakon 60 minuta bile su signifikantno niže u poređenju s rezultatima dobijenim unutar 15 minuta u arterijskim uzorcima krvi (po ±1.89 vs. 9.51±1.95; so ±5.03 vs ±4.5; p<0.01). Vrijednosti po 2 i so 2 u kapilarnim uzorcima također su bile signifikantno niže od vrijednosti dobijenih iz arterijskih uzoraka (po ±2.07 vs. 9.51±1.95; so ±4.86 vs ±4.50; p<0.01). Dobijene vrijednosti po 2 i so 2 u kapilarnim uzorcima u plastičnim kapilarnim cjevčicama bile su veće od onih u staklenim cjevčicama (po ±1.98 vs. 7.89±2.0; so ±11.04 vs ±11.22, p<0.01). Utjecaj na vrijednosti pco 2 u krvi nije bio signifikantan (p>0.05). Zaključak Vrijeme od uzimanja uzorka krvi do analiziranja, vrsta uzoraka i cjevčica za uzorkovanje imaju signifikantan učinak na parametre kiseonika u krvi. Analiza unutar 15 minuta od uzorkovanja smatra se prihvatljivom. Ključne riječi: analiza gasova u krvi, kapilarne cjevčice, šprice, staklo, plastika 112

11 ORIGINAL ARTICLE Association of LPIN1 gene variations with markers of metabolic syndrome in population from Bosnia and Herzegovina Tamer Bego 1, Tanja Dujić 1, Barbara Mlinar 2, Sabina Semiz 1,4, Maja Malenica 1, Besim Prnjavorac 3,6, Barbara Ostanek 2, Janja Marc 2, Anida Čaušević-Ramoševac 5, Adlija Čaušević 1 1 Department of Biochemistry and Clinical Analysis, Faculty of Pharmacy, University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 2 Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia, 3 General Hospital of Tešanj, Tešanj, 4 Faculty of Engineering and Natural Sciences, International University of Sarajevo, Sarajevo, 5 Bosnalijek, Pharmaceutical and Chemical Industry, Joint Stock Company, Sarajevo, 6 Department of Pathophysiology, Faculty of Pharmacy; University Sarajevo, Sarajevo; Bosnia and Herzegovina ABSTRACT Aim To investigate association of two LPIN1 gene variations with main traits of metabolic syndrome (MS) (waist circumference, body mass index, blood pressure, triglycerides, HDL-cholesterol and fasting glucose levels) in population from Bosnia and Herzegovina. Corresponding author: Tamer Bego Department for Biochemistry and Clinical Analysis, Faculty of Pharmacy, University of Sarajevo Zmaja od Bosne 8, Sarajevo, Bosnia and Herzegovina Phone: ; Fax: ; E.mail: tamer.bego@gmail.com Original submission: 28 November 2015; Revised submission: 03 February 2015; Accepted: 21 March doi: / Methods This study included 43 patients with metabolic syndrome and 43 healthy controls from General Hospital in Tešanj, Bosnia and Herzegovina. Subjects were genotyped for two LPIN1 gene variations (rs : C>T and rs : C>T) by real time PCR method. Results In control subjects LPIN1 polymorphism, rs : C>T, was significantly associated with a lower body mass index (BMI) (p=0.008) and waist circumference (p=0.008). The second analyzed rs : C>T polymorphism was associated with lower blood HbA1c levels (p=0.048) in a group of MS patients. Conclusion Results of our study suggest that rs : C>T polymorphism of LPIN1 gene could have a protective effect against development of metabolic syndrome, while rs : C>T might affect a glucose control in patients with MS. Keywords: metabolic syndrome, LPIN1 gene, markers, gene variations. Med Glas (Zenica) 2015; 12(2):

12 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION The metabolic syndrome (MS) is also known as syndrome X(1), the insulin resistance syndrome (2) and the deadly quartet (3). Metabolic syndrome is a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes (4). The pathogenesis of the metabolic syndrome is thought to involve a complex interaction of multiple factors, including obesity and abnormal fat distribution, insulin resistance, hepatic, vascular, and immunologic factors, as well as lifestyle and genetic contributions (4). The available evidence indicates that between 20% and 30% of the world s adult population have MS (5). New harmonized definition and criteria for MS was accepted finally in 2009, which included measurement of waist circumference, blood pressure, triglycerides, HDL-cholesterol and serum glucose (6). Insulin resistance and abdominal obesity appear to be predominant underlying risk factors of MS. Beside this, other associated conditions for this syndrome can be genetic factors, physical inactivity, aging, and hormonal imbalance (7). A newly discovered gene for lipin 1 (LPIN1) resides in the 2p25 region, and codes for phosphatidic acid phosphatase, a key enzyme in triglyceride (TG) biosynthesis (8). There is evidence that chromosome region 2p25 is in linkage disequilibrium with several obesity related phenotypes, such as body mass index (BMI), waist circumference, skin-fold thickness, and percentage of body fat (9). The lipin protein family consists of three members, lipin-1, lipin-2, and lipin-3 (10). Lipin 1 is a newly discovered multifunctional protein that participates in the metabolism of lipids in different ways (11). Lipin-1 is abundantly expressed in adipose tissue and skeletal muscle, and lipin-1 protein localizes to either the cytosol or the nucleus, which may be related to its two known functions (12). Namely, in the cytosol, lipin-1 acts as a phosphatidate phosphatase (PAP) enzyme converting phosphatidate to diacylglycerol during triglyceride biosynthesis (13-14), while in the nucleus of adipocytes and hepatocytes lipin-1 acts as a transcriptional coactivator that interacts with the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and PPARγ (PPARG) coactivator 1α (PPARGC1α) in a complex that modulates fatty acid oxidation gene expression (10, 15-16). Null mutations in the murine Lpin1 gene result in a severe defect in adipose tissue development, which is related to insulin resistance and fatty liver dystrophy (fld mice) (10). Based upon the comprehensive biological knowledge of lipin-1 role in energy metabolism, human lipin-1 has been considered as an obvious biological candidate to explain some of the inter-individual variations in the common metabolic phenotypes. In addition, variants in LPIN1 have been associated with the fasting serum insulin levels, body mass index (BMI), waist circumference, and obesity development (17-19). Previous data from four meta-analysis studies found an association of LPIN1 variants with increased BMI (20), while another study found an association with hypertension (21). Other studies have shown correlation of expression of LPIN1 gene in adipose tissue with BMI and insulin resistance in humans (14, 16). Recently published German population study (n=1674), showed an interesting association of LPIN1 gene variants with metabolic phenotype (22). They identified three associated three-marker haplotypes, one common haplotype that increased the risk for metabolic syndrome, while other two were associated with lower blood pressure levels, lower BMI, waist circumference, and HbA 1 C levels (22). In this study, for the first time in population from Bosnia and Herzegovina, it was analyzed whether LPIN1 gene polymorphisms (rs : C>T and rs : C>T), including haplotype analysis, were associated with the traits of metabolic syndrome. An association between biochemical parameters including, but not limited to, glucose, HbA1c, insulin levels, HDL and LDL cholesterol, triglycerides, serum proteins levels, and activity of liver enzymes and these two polymorphisms in LPIN1 gene was analyzed in patients with metabolic syndrome and healthy controls. PATIENTS AND METHODS Study participants The study included 43 patients with metabolic syndrome and 43 healthy controls from General Hospital in Tešanj, Zenica-Doboj Canton, Bosnia and Herzegovina. Investigation was done in accordance with ethical recommendations and practices of the General Hospital Tešanj, and with ethical 114

13 Bego et al. LPIN1 variations and metabolic syndrome principles outlined in the World Medical Association Declaration of Helsinki Ethical Principles of Medical Research Involving Human Subjects (initiated in June 1964, last amendment in October 2000). Each subject in the study signed written informed consent. Metabolic syndrome was diagnosed according to new harmonized definition and criteria for MS from According to this new definition, MS is diagnosed when any three of the following five criteria are met: increased waist circumference (recommended waist circumference thresholds for Euripides) 94 cm for men, and 80 cm for women), triglycerides 1.7 mmol/l, HDL-cholesterol < 1.0 mmol/l in males and < 1.3 mmol/l in females, blood pressure 130/85 mmhg and fasting glucose 5.6 mmol/l (6). Patients treated with insulin and patients with acute infection and / or inflammation and endocrine disorders were excluded from the study. All patients included in the study were using heterogeneous therapy (74% of all patients received antihypertensive therapy, 58% were treated with glucose-lowering drugs, and 47% were treated with lipid-lowering drugs). Healthy control group consisted of 43 non-obese, age-matched subjects, who had less than three features of MS. They were not taking any medication during the course of the study. Biochemical and anthropometrical measurements Waist circumference, height, weight, systolic and diastolic blood pressure were measured in all participants. BMI was calculated as weight (kg)/ (height (m)) 2. Serum levels of fasting glucose, triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, albumin, globulin, bilirubin, creatinine, urea, urate, HbA1c and C-reactive protein (CRP), as well as activities of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyltransferase were determined by using the VITROS auto analyzer 350 Chemistry System (Ortho-Clinical Diagnostics, Rochester, New York, USA). Serum insulin levels were measured by the Abbott AxSYM (Abbott Diagnostics, North Chicago, Illinois, USA) analyzer. HOMA IR index was calculated by using following formula: fasting insulin (mu/l) x fasting glucose (mmol/l)/22.5 (23). Genotyping analysis The Miller extraction protocol was used for the DNA extraction (24). Genotyping analysis for rs (IVS C > T, denoted intron 1 SNP) and rs (IVS17-228C > T, denoted intron 17 SNP) polymorphisms was performed with real-time PCR allelic discrimination on ABI PRISM with C_ _10 and C_ _10 assays, respectively (Applied Biosystems, Foster City, CA, USA). We double-genotyped twenty percent of all samples with 100% concordant results. Statistical analysis Chi-square (χ 2 ) and Fisher s exact tests (in the case where frequencies were less or equal to 5) were applied to examine differences in allele frequencies and genotype distributions between healthy controls and patients with MS. Significance of difference of biochemical and anthropometrical measurements according to genotypes of analyzed polymorphisms, sex and age were estimated by linear regression Corrections for Table 1. Characteristics of the study participants Parameter* MS patients (n=43) Controls (n=43) p Age (years) 49 (40-56) 45 (41-51) BMI (kg/m2) 33.0 ( ) 24.7 ( ) <0.001 Waist circumference (cm) 110 (96-120) 83 (78-90) <0.001 Systolic BP (mm Hg) 143 ( ) 120 ( ) <0.001 Diastolic BP (mm Hg) 90 (80-100) 78 (70-80) <0.001 Fasting insulin (mu/l) 10.6 ( ) 7.3 ( ) Fasting glucose (mmol/l) 8.4 ( ) 5.0 ( ) <0.001 HOMA-IR 4.1 ( ) 1.6 ( ) <0.001 Blood HbA1c (%) 6.1 ( ) 5.6 ( ) <0.001 Total cholesterol (mmol/l) 5.6 ( ) 5.8 ( ) LDL-cholesterol (mmol/l) 3.20 ( ) 3.37 ( ) HDL-cholesterol (mmol/l) 1.07 ( ) 1.67 ( ) <0.001 Triglycerides (mmol/l) 2.26 ( ) 1.17 ( ) <0.001 CRP (mg/l) 5.0 ( ) 1.3 ( ) <0.001 Creatinine (mmol/l) 82.5 ( ) 96.0 ( ) <0.001 Urea (mmol/l) 5.05 ( ) 4.30 ( ) Urate (mmol/l) ( ) ( ) Albumin (g/l) 49.4 ( ) 44.0 ( ) Globulin (g/l) 24.6 ( ) 32.0 ( ) <0.001 Bilirubin (mmol/l) 12.9 ( ) 11.5 ( ) AST (IU/L) 26.0 ( ) 26.0 ( ) ALT (IU/L) 27.0 ( ) 23.0 ( ) GGT (IU/L) 23.5 ( ) 17.5 ( ) *Values represent medians (lower-upper quartile); significance of difference in Mann-Whitney test. BMI, body mass index; BP, blood pressure; HOMA-IR, homeostasis model assessment insulin resistance index; LDL, low-density lipoprotein; HDL, high-density lipoprotein; hscrp, high-sensitivity C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT γ, glutamyl transferase γ 115

14 Medicinski Glasnik, Volume 12, Number 2, August 2015 multiple testing were performed by using Bonferroni correction for a total of two SNPs. A p value 0.05 was considered statistically significant. Linkage disequilibrium was calculated by using the 2LD program (25). Haplotype reconstruction was done with the PHASE program (26). RESULTS All analyzed parameters were significantly different between patients and control groups except total cholesterol, LDL cholesterol, and bilirubin levels, as well as aspartate aminotransferase (AST) and γ-glutamyltransferase activity (GGT) (Table 1). Parameter* BMI (kg/m2) Waist circumference (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Fasting insulin (mu/l) Fasting glucose (mmol/l) HOMA-IR Blood HbA1c (%) Total cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triglycerides (mmol/l) hscrp (mg/l) Creatinine (mmol/l) Urea (mmol/l) Urate (mmol/l) Albumin (g/l) Globulin (g/l) Bilirubin (mmol/l) AST (IU/L) ALT (IU/L) GGT (IU/L) Table 2. Allele and genotype frequencies for LPIN1 gene polymorphisms* Polymorphism Intron 1SNP (rs ) Intron 17 SNP (rs ) Table 3. Effects of LPIN1 SNPs on biochemical and anthropometrical parameters in controls C/C (n=14) 23.3 ( ) 81 (72-85) 120 ( ) 75 (66-87) 7.3 ( ) 5.1 ( ) 1.6 ( ) 5.6 ( ) 6.2 ( ) 3.81 ( ) 1.72 ( ) 1.15 ( ) 1.0 ( ) 63.0 ( ) 4.5 ( ) ( ) 44.5 ( ) 30.0 ( ) 9.6 ( ) 22.0 ( ) 21.0 ( ) 16.0 ( ) rs C>T C/T + T/T (n=24) B (95% CI) p pb ( ) (-0.758, 5.071) (78-93) (-1.405, ) ( ) (-8.272, ) (75-80) (-5.21, ) ( ) (-7.582, 0.290) ( ) (-0.392, 0.213) ( ) (-1.905, 0.144) ( ) (-0.289, 0.611) ( ) (-1-059, 0.392) ( ) (-0.904, 0.596) ( ) (-0.474, 0.064) ( ) (-0.439, 0.294) ( ) (-0.582, ( ) (-3.075, ) ( ) (-0.70, ) ( ) ( , 44,634) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) T-allele MS frequency CC 16 (40.0%) 0.36 Controls 14 (33.3%) CT 19 (47.5%) 23 (54.8%) TT 5 (12.5%) 5 (11.9%) Total p CC 26 (65.0%) 32 (76.2%) CT 12 (30.0%) (21.4%) TT 2 (5.0%) 1 (2.4%) Total p T-allele frequency p 0, *rs : C>T and rs : C>T; Significance of χ2 / Fisher s exact test for comparison of genotype frequencies between healthy controls and MS patients; p value for Hardy-Weinberg equilibrium. C/C (n=29) 25.4 ( /.7) 86 (80-93) 120 ( ) 80 (70-80) 6.9 ( ) 5.0 ( ) 1.5 ( ) 5.6 ( ) 5.9 ( ) 3.34 ( ) 1.69 ( ) 1.13 ( ) 1.5 ( ) 70.0 ( ) 4.7 ( ) 285 ( ) 44.0 ( ) 32.0 ( ) 11.7 ( ) 26.0 ( ) 23.0 ( ) 17.0 ( ) rs C>T C/T + T/T (n=10) B (95% CI) p pb ( ) (-7.104, ) (72-81) ( , ) ( ) ( , 7.004) (67-82) (-8.974, 7.026) ( ) (-6.112, 2.722) ( ) (-0.606, 0.010) ( ) (-1.697, 0.564) ( ) (-0.420, 0.549) ( ) (-0.584, 0.981) ( ) (-0.679, 0.926) ( ) (-0.283, 0.312) ( ) (-0.401, 0.385) ( ) (-2.890, 0.853) ( ) ( , ) ( ) (-1.724, 0.106) ( ) ( , ) ( ) (-2.302, 3.540) ( ) (-4.062, 3.913) ( ) (-4.578, 1.777) ( ) (-5.399, 3.105) ( ) (-3.894, ) ( ) (-1.500, ) *Values represent medians (lower-upper quartile); Effects (unstandardized coefficients B) and p values were assessed using multiple linear regression adjusted for age and gender, under dominant genetic model; pb, adjusted by using Bonferroni correction for two SNPs; BMI, body mass index; BP; blood pressure; HOMA-IR, homeostasis model assessment insulin resistance index; LDL, low-density lipoprotein; HDL, high-density lipoprotein; hscrp, high-sensitivity C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT-γ; glutamyltransferase; 116

15 Bego et al. LPIN1 variations and metabolic syndrome Table 4. Effects of LPIN1 SNPs on biochemical and anthropometrical parameters in the patients Parameter* BMI (kg/m2) Waist circumference (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg) Fasting insulin (mu/l) Fasting glucose (mmol/l) HOMA-IR Blood HbA1c (%) Total cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triglycerides (mmol/l) hscrp (mg/l) Creatinine (mmol/l) Urea (mmol/l) Urate (mmol/l) Albumin (g/l) Globulin (g/l) Bilirubin (mmol/l) AST (IU/L) ALT (IU/L) GGT (IU/L) rs C>T rs C>T C/C C/T + T/T pb C/C C/T + T/T B (95% CI) p (n=16) (n=24) (n=26) (n=14) B (95% CI) p pb ( ) ( ) (-3.845, 4.404) ( ) ( ) (-5.904, 2.253) ( ) (96-123) ( , 6.659) ( ) (87-122) ( , 5.746) ( ) ( ) ( , 8.317) ( ) ( ) ( , 6.392) (81-100) (80-97) ( , 4.647) (81-100) (70-97) ( , 2.277) ( ) ( ) (-4.951, 5.712) ( ) ( ) (-6.191, 4.454) ( ) ( ) (-5.305, 0.675) ( ) ( ) (-5.124, 0.888) ( ) ( ) (-1.909, 2.084) ( ) ( ) (-3.006, 0.912) ( ) ( ) (-2.010, ) ( ) ( ) (-1.222, 0.775) ( ) ( ) (-0.629, 0.913) ( ) ( ) (-0.996, 0.549) ( ) ( ) (-0.515, 1.134) ( ) ( ) (-0.848, 0.815) ( ) ( ) (-0.181, 0.266) ( ) ( ) (-0.438, ) ( ) ( ) (-1.473, 0.283) ( ) ( ) (-1.013, 0.788) ( ) ( ) (-1.946, 1.740) ( ) ( ) (-1.608, 2.074) ( ) ( ) ( , ) ( ) ( ) ( , ) ( ) (4.66.2) (-0.703, 1.590) ( ) ( ) (-1.266, 1.047) ( ) ( ) ( , ) ( ) ( ) ( , ) ( ) ( ) (-4.794, 3.195) ( ) ( ) (-6.488, 1.299) ( ) ( ) (-4.852, 2.746) ( ) ( ) (-4.277, 3.350) ( ) ( ) (-5.738, 0.921) ( ) ( ) (-5.993, 0.613) ( ) ( ) (-4.941, ) ( ) ( ) ( , 2.686) ( ) ( ) ( , ) ( ) ( ) ( , ) ( ) ( ) ( , ) ( ) ( ) ( , 4.721) *Values represent medians (lower-upper quartile); Effects (unstandardized coefficients B) and p values were assessed using multiple linear regression adjusted for age and gender, under dominant genetic model; pb adjusted by using Bonferroni correction for two SNPs; BMI, body mass index; BP; blood pressure; HOMA-IR, homeostasis model assessment insulin resistance index; LDL, low-density lipoprotein; HDL, high-density lipoprotein; hscrp, high-sensitivity C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT-γ; glutamyltransferase; Table 5. Haplotype frequencies and diplotypes of LPIN1 gene in MS patients and healthy controls Haplotype (intron 1 SNP-intron MS 17 SNP)* patients Frequency Controls Diplotype Diplotype No No. of subjects MS patients Controls CC CC/CC CT CC/CT TC CC/TC TT CC/TT CT/CT 5-1 CT/TC 6-3 CT/TT TC/TC TC/TT *corresponding to the rs : C>T (intron SNP1) and rs : C>T (intron SNP17) polymorphism Allele frequencies were in Hardy-Weinberg equilibrium for both, patients and control subjects (p>0.05). However, no significant differences in analyzed genotype frequencies were found between patients and healthy controls (Table 2). In a control group, after Bonferroni correction, rs : C>T polymorphism did not show significant association with any biochemical or anthropometrical measurements. However, the carriers of T allele (CT + TT) of another analyzed polymorphism of LPIN1 gene, rs : C>T, had significantly lower BMI (p=0,008), waist circumference (p=0.008), and tendency of association with lower 117

16 Medicinski Glasnik, Volume 12, Number 2, August 2015 creatinine levels (p=0.058), as compared to the carriers of a wild type allele (CC) (Table 3). In group of MS patients, the carriers of T allele (CT + TT) of rs : C>T polymorphism had significantly lower blood HbA 1c (%) (p=0.048), as compared to the carriers of a wild type allele (CC). The carriers of T allele (CT + TT) of rs : C>T polymorphism had significant lower ALT activity (p=0.012) and tendency of association with lower HDL cholesterol levels (p=0.066), as compared to the carriers of a wild type allele (CC). As shown in Table 4, no association was found for both analyzed LPIN1 gene polymorphisms with anthropometrical measurements (body mass index (BMI) and waist circumference). The selected LPIN1 variants, rs : C>T and rs : C>T were in a weak linkage disequilibrium (D =0.261). No significant differences in distribution of haplotype frequencies between patients with MS and control subjects were demonstrated. Furthermore, an association of LPIN1 haplotypes with biochemical and anthropometrical parameters was also tested. In control group, the carriers of CC haplotype had significantly higher plasma insulin (p=0.024), higher glucose levels (p=0.036) and higher HOMA IR index (p=0.024) as compared to the carriers of CT haplotype. No significant associations of LPIN1 haplotypes with traits of MS were found in patients group (Table 5). DISCUSSION Members of the lipin protein family have a newly discovered enzymatic role in triglyceride and phospholipid biosynthesis as a phosphatidate phosphatase, and act also as inducible transcriptional coactivators in conjunction with peroxisome proliferator-activated receptor c (PPARc) coactivator-1a and PPARa (13). Through these activities, the founding member of the family, lipin-1, influences lipid metabolism and glucose homeostasis (13). Results of our study showed significant association of rs : C>T genetic variant with body mass index (BMI) and waist circumference. No significant associations between disease-associated traits and rs : C>T were found. Since the majority of patients participating in this study were using medications (antihypertensive, glucose-lowering or lipid-lowering drugs), the influence of LPIN1 gene polymorphisms on the most of selected biochemical parameters in MS patients should be interpreted cautiously. However, it was reasonable to analyze the effects of LPIN1 gene variations on the BMI and waist circumference in these patients. The selected genotype-phenotype analysis showed that, the mutant T allele of rs : C>T polymorphism was associated with lower blood HbA 1 in a group of MS patients. T allele of another analyzed polymorphism in our study, rs : C>T, was associated with lower BMI and waist circumference, and creatinine levels in controls, and lower HDL cholesterol levels and lower ALT activity in a group of MS patients. Only a few studies analyzed association of LPIN1 gene polymorphisms (rs : C>T and rs : C>T) with metabolic syndrome (17,22,27). Suviolahti et al. analyzed seven polymorphisms in the LPIN1 gene. They found an association of rs : C>T polymorphism with the insulin levels. In addition, rs : C>T and rs : C>T polymorphism were associated with BMI in lean males. Since an association of LPIN1 variants with BMI in the lean or obese females was not found, the association of LPIN1 alleles with BMI appeared to be sex specific (17). Mlinar et al., in their study tested an association of LPIN1 polymorphisms (rs : C>T and rs : C>T) with polycystic ovary syndrome (PCOS) (28). Their results showed that mutated T allele of rs : C>T polymorphism was associated with lower plasma LDL-cholesterol levels in controls, with lower glucose levels after OGTT in the PCOS patients, and with lower insulin levels and HOMA-IR in nonobese PCOS patients. These results suggest a protective role of mutated T allele of rs : C>T polymorphism against development of IR and dyslipidemia. Mutated allele T of another analyzed polymorphism in this study (28), rs : C>T, showed an association with higher triglyceride levels in control subjects, suggesting a negative effect of this polymorphism on the metabolic phenotype. Wiedmann at al. in their study analyzed an association of 15 genetic variants of LPIN1 gene, including rs : C>T, with metabolic phenotype. Their results showed the borderline significant 118

17 Bego et al. LPIN1 variations and metabolic syndrome REFERENCE 1. Reaven GM. Banting lecture Role of insulin resistance in human disease. Diabetes 1988; 37: DeFronzo RA, Ferrannini E. Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991; 14: association of this polymorphism (rs : C>T) with higher plasma triglyceride levels (22). In our recent study we have also tested an association of LPIN1 and PPARG variants with biochemical and anthropometrical parameters in patients with MS and type 2 diabetes. Interestingly, results of this study showed that mutated T allele of rs : C>T polymorphism was associated with higher insulin levels in patients with MS and type 2 diabetes (27). Neither of these studies analyzed an association of two LPIN1 variants (rs : C>T and rs : C>T) with the biochemical parameters, including albumin, globulin, creatinine, urea and uric acid levels, as well as ALT, AST and GGT activity that we tested in the current study. The mechanism of an association of LPIN1 polymorphisms with above biochemical parameters is not completely understood. However, many previous studies demonstrated an association of selected biochemical parameters, which are known as possible markers, with increased risk of development Type 2 diabetes (T2D) (29-33). Recently published studies found a positive correlation between higher creatinine and urea levels with increased risk for T2D development (32-33). Furthermore, other studies found a correlation of higher activity of liver enzymes (AST, ALT or GGT) with increased risk for MS and T2D development, or their association with obesity and insulin resistance (29-31). Effects of two polymorphisms of LPIN1 gene (rs : C>T and rs : C>T) on metabolic phenotype was further tested by the haplotype analyses. An association of rs : C>T polymorphism with the lower BMI and waist circumference, and protective role of this polymorphism were confirmed by this analysis. Another analyzed polymorphism, rs : C>T, did not show any association disease-associated traits, that is also confirmed by haplotype analysis. In control subjects, carriers of CT haplotype had significant lower insulin levels, glucose levels and HOMA IR levels as compared to the carriers of CC haplotype. Thus, these findings suggested that non-mutated first locus, and mutated second locus (CT haplotype) decreased metabolic risk, while non-mutated first and second locus (CC haplotype) increased metabolic risk. These positive findings have to be replicated in a larger cohort of subjects. In conclusion, the reason why we have chosen these two SNPs of LPIN1 gene is because previous studies have shown association with metabolic phenotype, but with opposite results, prompting us to examine impact of these polymorphisms of LPIN1 gene in development of metabolic syndrome in the population of Bosnia and Herzegovina. These two polymorphisms did not cover the role of all variation of LPIN1 gene, which is a limitation of our study. Results of our study showed that rs : C>T decrease the risk of MS, while rs : C>T did not show any association with risk factors of MS. An association of rs : C>T polymorphism with lower BMI and waist circumference suggest that this genetic variant of LPIN1 gene could have a protective role against development of metabolic syndrome. Future investigations including larger cohorts of subjects, genes and larger number of SNPs, as well as studies on other populations, are needed to substantiate these findings. Illuminating the mechanism and pathogenesis of this complex disorder, might lead to effective treatment, and also to predict individual s risk of developing of metabolic syndrome. ACKNOWLEDGEMENTS We greatly appreciate the technical assistance of Mr. Nermin Kotoric, Mr. Hajrudin Mujic and Ms. Elma Topić. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare 3. Kaplan NM. The deadly quartet. Upper-body obesity, glucose intolerance, hypertriglyceridemia, and hypertension. Arch Intern Med 1989; 149: Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet 2005; 365: Grundy SM. Metabolic syndrome pandemic. Arterioscler Thromb Vasc Biol 2008; 28:

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19 Bego et al. LPIN1 variations and metabolic syndrome Povezanost varijacija LPIN1 gena s markerima metaboličkog sindroma u populaciji Bosne i Hercegovine Tamer Bego 1, Tanja Dujić 1, Barbara Mlinar 2, Sabina Semiz 1,4, Maja Malenica 1, Besim Prnjavorac 3,6, Barbara Ostanek 2, Janja Marc 2, Anida Čaušević-Ramoševac 5, Adlija Čaušević 1 1 Katedra za biohemiju i kliničke analize, Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo, Bosna i Hercegovina; 2 Katedra za kliničku biohemiju, Farmaceutski fakultet, Univerzitet u Ljubljani, Ljubljana, Slovenija; 3 Opća bolnica u Tešnju, Tešanj, 4 Fakultet za inženjering i prirodne nauke, Internacionalni univerzitet u Sarajevu, Sarajevo, 5 Bosnalijek d. d, Farmaceutska i hemijska industrija, Sarajevo, 6 Katedra za patofiziologiju, Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo; Bosna i Hercegovina SAŽETAK Cilj Istražiti povezanost dvije varijante LPIN1 gena s glavnim karakteristikama metaboličkog sindroma (MS) (opseg struka, indeks tjelesne mase, krvni pritisak, trigliceridi, HDL holesterol i glukoza) u populaciji Bosne i Hercegovine. Metode Studija je uključila 43 pacijenta s metaboličkim sindromom i 43 zdrava ispitanika (kontrole) iz Opće bolnice u Tešnju. Varijante LPN1 gena (rs : C>T i rs : C>T) analizirane su PCR metodom (real time PCR). Rezultati Kod kontrolnih ispitanika, polimorfizam LPIN1 gena, rs : C>T, sa statistički značajnom razlikom bio je povezan s nižim vrijednostima indeksa tjelesne mase (ITM) (p=0.008) i opsega struka (p=0.008). Drugi analizirani genski polimorfizam, rs : C>T, pokazao je povezanost s nižim vrijednostima HbA1c (p=0.048) u skupini pacijenata s MS-om. Zaključak Rezultati studije sugerišu da bi rs : C>T polimorfizam LPIN1 gena mogao imati zaštitnu ulogu u razvoju metaboličkog sindroma, dok bi polimorfizam rs : C>T mogao imati ulogu u kontroli glukoze kod pacijenata s MS-om. Ključne riječi: metabolički sindrom, LPIN1 gen, marker, varijacije gena 121

20 ORIGINAL ARTICLE Alpha-lipoic acid reduces body weight and regulates triglycerides in obese patients with diabetes mellitus Azra Okanović 1, Besim Prnjavorac 2,3, Edin Jusufović 4,5, Rifat Sejdinović 2, 6 1 Health Centre Tešanj, 2 Department for Internal and Lung Diseases of General Hospital Tešanj, 3 Pharmaceutical Faculty of University in Sarajevo, 4 Polyclinic for Pulmonary Diseases of Health and Educational Institution Dr. Mustafa Šehović Tuzla, 5 Medical School of University in Tuzla, 6 Faculty of Health Care, University in Zenica; Bosnia and Herzegovina ABSTRACT Aim To determine an influence of alpha-lipoic acid to reduction of body weight and regulation of total cholesterol concentration, triglycerides and glucose serum levels in obese patients with diabetes mellitus type 2. Corresponding author: Besim Prnjavorac Department of Internal and Lung disease, General Hospital Tešanj Braće Pobrića 17, Tešanj, Bosnia and Herzegovina Phone: ; Fax: ; pbesim@bih.net.ba Methods A prospective study includes two groups of obese patients with diabetes mellitus and signs of peripheral polyneuropathia: examined group (30 patients; 15 females and 15 males), and control group (30 patients; 12 females and 18 males). All were treated with metformin ( mg/day). Examined patients were additionally treated with alpha-lipoic acid 600 mg/day during 20 weeks. Body mass index and concentrations of total cholesterol, triglycerides and glucose in serum were compared before and after the treatment. Results The group treated with 600 mg alpha-lipoic acid lost significantly more weight, and had lower triglyceride level than the control group. There were no significant differences in total cholesterol and glucose serum levels between the groups. Conclusion Alpha-lipoic acid of 600 mg/day treatment have influenced weight and triglycerides loss in obese patients with diabetes mellitus type 2. It should be considered as an important additive therapy in obese patients with diabetes mellitus type 2. Key words: body mass index, serum glucose, lipid status. Original submission: 28 November 2014; Revised submission: 06 July 2015; Accepted: 09 July doi: / Med Glas (Zenica) 2015; 12(2):

21 Okanović et al. Alpha-lipoic acid in treatment of diabetes mellitus INTRODUCTION Most patients with diabetes mellitus type 2 suffer from disorders of lipoprotein metabolism as well as obesity (1). Diabetic dyslipoproteinemia is characterised with increased levels of total cholesterol, low density lipoproteins (LDL) and triglycerides, as well as decreased level of high density lipoproteins (HDL) (1-3). Lipid metabolic changes and obesity are both strong and intensive risk factors for developing complications, first of all microvascular ones (1,4). Therefore, it is very important to correct properly metabolism disorders of lipoprotein and obesity in diabetes mellitus type 2 patients (1,3). Alpha lipoic acid is established in many studies as a protector against oxidative damage cells, which is described in diabetes mellitus patients (5) and positively influences the regulation level of glucose (6) and reduces blood lipids (total cholesterol, LDL, and triglycerides (7). It has been shown that that alpha-lipoic acid markedly reduces body weight gain in rodents (8), but also in humans (9-11). Also, alpha-lipoic acid has shown to be effective in reducing symptoms of diabetic polyneuropathy without serious adverse effects (12,13). The usual daily intake of alpha lipoic acid by foods (muscles, heart, kidney, liver) is quite low in order to achieve a therapeutic effect in conditions of increased needs for this substance. (10). Therefore, the potential health implications of alpha lipoic acid have been investigated in clinical practice in countries such as Germany and Korea (14) with multicentric trials currently ongoing in Europe and North America (15). These studies have included products containing alpha lipoic acid in a very wide range of doses, ranging mg/day (2,6, 9-13, 16-19). The aim of this study was to examine the effect of alpha lipoic acid in reducing body weight, the regulation of lipid status, as well as the regulation of glucose in blood in obese patients with diabetes mellitus type 2. PATIENTS AND METHODS This prospective study has been done in Public Health Centre Tešanj, Bosnia and Herzegovina, in the period from May to September Sixteen obese patients with diabetes mellitus type 2 and signs of peripheral neuropathy were included. All were treated with metformin (850 to 1700 mg/day) and divided into 2 groups: examined (30; 25 females and 5 males) and control (30; 22 females and 8 males). Control patients were additionally treated with alpha-lipoic acid of 600 mg/day during 20 weeks. Body mass index and serum concentrations of glucose, cholesterol and triglycerides were measured and compared before and after the treatment, as well as between examined and control group. Body mass index was calculated from body weight and height according to the formula: BMI (kg/m 2 ) = Weight (kg) / Height (m) 2. Referral values of observed parameters were: body mass index , concentration of glucose mmol/l, concentration of cholesterol mmol/l and concentration of triglycerides mmol/l. The distribution of values was determined by D Agostino test. Mean values were shown as mean ± standard deviation. Student s t-test, Mann-Whitney test, Fisher s test and χ 2 test, with double and single orientation, are used for calculating the difference between the groups. ANOVA test was used to calculate relative differences of variance of the distribution between the variables. Statistical hypotheses were tested at the level of α=0.05, and the difference between the groups was considered significant if p<0.05 or less. RESULTS Sex distribution was similar in both groups (p=0.6042). Age distribution was similar among patients between the groups for both genders, as well as after stratification to male and female patients. Also, age distribution was similar among male and female patients in both groups (experimental group: p=0.1691; control group: p=0.4541) (Table 1). Table 1. Age and sex distribution in experimental and control group Gender No (%) of patients Control Experimental Mean age ± standard deviation Experimental Control Males 15 (50) 18 (60) 64.40± ± Females 15 (50) 12 (40) 61.53± ± Total 30 (100) 30 (100) 62.97± ± Before the treatment, body mass index in both groups was similar (p>0.05). p 123

22 Medicinski Glasnik, Volume 12, Number 2, August 2015 Body mass index was significantly lower in experimental and control groups after the treatment (p<0.001 and p=0.01, respectively). Body mass index was significantly lower in the group treated with metformin and alpha-lipoic than in the group treated with metformin only (p<0.05) (Figure 1). Despite lowering of serum concentration of total cholesterol after the treatment, differences were not significant before and after the treatment in any of the two groups (p>0.5 ). Also, despite lower concentration of total cholesterol after the treatment in the group treated with metformin and alpha-lipoic acid than in the group treated with metformin only, this difference was not significant (p>0.05) Before, as well as after the treatment, serum concentration of glucose was similar in both groups (p>0.05). After the treatment serum concentration of glucose was significantly lower compared to concentration before the treatment in both groups (p<0.001) (Figure 3). Figure 1. Body mass index in experimental and control group before and after the treatment Serum concentration of triglycerides was similar in both groups before the treatment (p>0.05). After the serum concentration of triglycerides was significantly lower in both groups (experimental group: p<0.01; control group: p<0.05). After the treatment, serum concentration of triglycerides was significantly lower in the group treated with metformin and alpha-lipoic than the group treated with metformin only (p<0.5) (Figure 2). Figure 3. Serum concentration of glucose in experimental and control group before and after the observed period Achievement referral values of body mass index, concentration of tryglicerides, as well as glucose, before and after the treatment were similar (p=0.0562, p= and p=0.5, respectively) (Figure 4). Figure 2. Serum concentration of triglycerides in experimental and control group before and after the observed period Before and after the treatment, serum concentration of total cholesterol was similar in both groups (p>0.5). Figure 4. Achievement referral values (Normalisation) of body mass index (A), serum concentration of triglycerides (B) and serum concentration of glucose (C) after the treatment in experimental and control group. DISCUSSION There has been little research that would evaluate an effect of oral treatment of humans with diabetes mellitus type 2 with alpha lipoic acid so far 124

23 Okanović et al. Alpha-lipoic acid in treatment of diabetes mellitus (2,9,10). Limited evidence from human studies suggests that alpha lipoic acid may be an effective body weight or lipid-lowering compound (20). To our knowledge, this is the first study in Bosnia and Herzegovina showing that alpha-lipoic acid treatment could lead to a significant weight reduction and regulate lipid status in obese patients with diabetes mellitus type 2. As a dietary supplement, alpha lipoic acid appears to have broad molecular specificity with an impressive array of metabolic benefits including protection against weight gain (10), diet-induced dyslipidemia (4,7), arterial lesion formation (5,18,19), and insulin resistance (21). Our research included patients in experimental and control group with similar sex and age distribution, which led to a minimization of impact of these factors on metabolic and oxidative changes that could be possibly linked to sex differences or aging. Several studies showed body weight loss and reduction of triglycerides serum concentration in obese patients with diabetes mellitus type 2 treated with alpha lipoic acid (4,9, 10-12). But, some of the studies showed that oral dose of 600 mg/ day of alpha lipoic acid did not influence weight lost or normalization of lipid status (9). However, the dose of 600 mg/day applied intravenously led to a significant reduction in plasma free fatty acids, triglycerides, total cholesterol, LDL-cholesterol, oxidized LDL-cholesterol, and VLDLcholesterol in obese patients treated for 2 weeks (21). Therefore, in most studies the patients were treated with 1200 mg/day or higher orally (2,6, 9-12, 16,18). In the randomized double-blind and placebo-controlled study during 20 weeks 360 obese individuals (body mass index kg/m2 plus hypertension, diabetes mellitus, or hypercholesterolemia) were randomized to alpha-lipoic acid 1200 or 1800 mg/day or placebo. Reduction in body mass index was significantly greater in the 1800 mg/day alpha-lipoic acid group than in the placebo group, as was the percentage of patients who achieved a 5% reduction in baseline body weight (21.6% vs 10.0%) (9). In our research, patients who completed 20 weeks treatment with 600 mg/day of alpha-lipoic acid orally showed modest but significant reduction in body mass index and serum triglycerides concentration in comparison with the patients that were not treated with alpha lipoic acid. Hence our results are opposite to literature results with regard to orally administrated alpha-lipoic acid dose. However, 20 weeks treatment in our research was longer than in other studies and this might result in regulation of body weight and triglycerides. At the same time, after the observed period there were no differences in frequencies of normalized weight or triglyceride between the group treated with and group not treated with alpha-lipoic acid in our research. Considering literature studies (2,9,12,13,18) and results of our study, doses of alpha-lipoic acid higher than 600 mg/day could have more benefit in obese patients with diabetes mellitus type 2. Although the maximum tolerated dose of alpha-lipoic acid in human patients has not been well defined, some studies have suggested that humans can tolerate several grams per day of oral alpha-lipoic acid (9). Thus, this could be a part of another prospective research based on tolerance of alpha-lipoic acid. In the majority of studies alpha-lipoic acid led to significant reduction of serum total cholesterol concentration in obese patients (3,4, 6-9). In our research the cholesterol concentration did not differ after the treatment with alpha-lipoic acid in comparison with baseline level at the beginning of the treatment. Possible reasons for this could be that the patients in our study were treated with 600 mg/day, but in majority of other studies this dose was pretty higher (9,10, 12-14, 18,21). Also considering low dose of alpha-lipoic acid, the treatment period (20 weeks) was relatively short in our study. Although alpha lipoic acid regulates glucose concentration (2,6,8) in our study there were no differences between groups in glucose concentration after the treatment with alpha-lipoic acid. This could be explained with the fact that metformin, given in both groups, leads to a significant reduction of glucose levels in obese patients (22), as well as relatively low dose (600 mg/day) of alpha-lipoic acid; probable reason is a relatively short treatment period. This research has several limitations. The duration of observed period (20 weeks) was relatively short. Furthermore, outpatients were not monitored intensively with regard to individual conducting of hypocaloric diet, which was prescribed in a similar way to all patients. 125

24 Medicinski Glasnik, Volume 12, Number 2, August 2015 In conclusion, this study showed that 600 mg/day of oral alpha-lipoic acid was effective in achieving significant regulation of weight loss and serum triglycerides in obese patients with diabetes mellitus type 2, but without difference in frequencies of normalization in patients with and without alpha-lipoic acid. Alpha-lipoic acid may be effective as an additional treatment in obese patients with diabetes mellitus type 2, but further studies are required to determine an adequate dosage of alpha-lipoic acid as well as long-term safety and efficacy. ACKNOWLEDGEMENT Authors would like to thank all colleagues from Health Centre Tešanj, Bosnia and Herzegovina who supported us and took part in collecting all necessary materials needed for this study. Also, thanks to all patients who accepted to be part of the study. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. REFERENCES: 1. Shin JA, Lee JH, Lim SY, Ha HS, Kwon HS, Park YM, Lee WC, Kang MI, Yim HW, Yoon KH, Son HY. Metabolic syndrome as a predictor of type 2 diabetes, and its clinical interpretations and usefulness. J Diabetes Investig 2013; 4: Porasuphatana S, Suddee S, Nartnampong A, Konsil J, Harnwong B, Santaweesuk A. Glycemic and oxidative status of patients with type 2 diabetes mellitus following oral administration of alpha-lipoic acid: a randomized double-blinded placebo-controlled study. Asia Pac J Clin Nutr 2012; 21: Choi SH, Ginsberg HN. Increased very low density lipoprotein (VLDL) secretion, hepatic steatosis, and insulin resistance. Trends Endocrinol Metab 2011; 22: Seo EY, Ha AW, Kim WK. Alpha-Lipoic acid reduced weight gain and improved the lipid profile in rats fed with high fat diet. Nutr Res Pract 2012; 6: Gupta S, Gambhir JK, Kalra O, Gautam A, Shukla K, Mehndiratta M, Agarwal S, Shukla R. Association of biomarkers of inflammation and oxidative stress with the risk of chronic kidney disease in Type 2 diabetes mellitus in North Indian population. J Diabetes Complications 2013; 27: MorakinyoAO, Awobajo FO, Adegoke OA. Effects of alpha lipoic acid on blood lipids, renal indices, antioxidant enzymes, insulin and glucose level in streptozotocin-diabetic rats. Biology and Medicine 2013; 5: Kandeil MA, Amin KA, Hassanin KA, Ali KM, Mohammed ET. Role of lipoic acid on insulin resistance and leptin in experimentally diabetic rats. J Diabetes Complications 2011; 25: Song KH, Lee WJ, Koh JM, Kim HS, Youn JY, Park HS, Koh EH, Kim MS, Youn JH, Lee KU, Park JY. Alpha-Lipoic acid prevents diabetes mellitus in diabetes-prone obese rats. Biochem Biophys Res Commun 2005; 326: Koh EH, Lee WJ, Lee SA, Kim EH, Cho EH, Jeong E, Kim DW, Kim MS, Park JY, Park KG, Lee HJ, Lee IK, Lim S, Jang HC, Lee KH, Lee KU. Effects of alpha-lipoic acid on body weight in obese subjects. Am J Med 2011; 124: Carrier B, Rideout TC. Anti-Obesity and Lipid- Lowering Properties of Alpha-Lipoic Acid J Hum Nutr Food Sci 2013; 1: Ratliff JC, Palmese LB, Reutenauer EL, Tek C. An open-label pilot trial of alpha-lipoic acid for weight loss in patients with schizophrenia without diabetes. Clin Schizophr Relat Psychoses 2013; 7: Han T, Bai J, Liu W, Hu Y. A systematic review and meta-analysis of α-lipoic acid in the treatment of diabetic peripheral neuropathy. Eur J Endocrinol 2012; 167: Xu Q, Pan J, Yu J, Liu X, Liu L, Zuo X, Wu P, Deng H, Zhang J, Ji A. Meta-analysis of methylcobalamin alone and in combination with lipoic acid in patients with diabetic peripheral neuropathy. Diabetes Res Clin Pract 2013; 101: Hahm JR, Kim BJ, Kim KW. Clinical experience with thioctacid (thioctic acid) in the treatment of distal symmetric polyneuropathy in Korean diabetic patients. J Diabetes Complications 2004; 18: Ziegler D. Thioctic acid for patients with symptomatic diabetic polyneuropathy: a critical review. Treat Endocrinol 2004; 3: Gębka A, Serkies-Minuth E, Raczyńska D. Effect of the administration of alpha-lipoic acid on contrast sensitivity in patients with type 1 and type 2 diabetes. Mediators Inflamm 2014; 2014: Lee WR, Kim A, Kim KS, Park YY, Park JH, Kim KH, Kim SJ, Park KK. Alpha-lipoic acid attenuates atherosclerotic lesions and inhibits proliferation of vascular smooth muscle cells through targeting of the Ras/MEK/ERK signaling pathway. Mol Biol Rep 2012; 39: Sun YD, Dong YD, Fan R, Zhai LL, Bai YL, Jia LH. Effect of alpha lipoic acid supplementation on serum lipids and antioxidative ability in patients with agerelated macular degeneration. Ann Nutr Metab 2012; 60: Cicek M, Yıldırır A, Okyay K, Yazici AC, Aydinalp A, Kanyilmaz S, Muderrisoglu H. Use of alpha-lipoic acid in prevention of contrast-induced nephropathy in diabetic patients. Ren Fail 2013; 35:

25 Okanović et al. Alpha-lipoic acid in treatment of diabetes mellitus 20. Chen WL, Kang CH, Wang SG, Lee HM. α-lipoic acid regulates lipid metabolism through induction of sirtuin 1 (SIRT1) and activation of AMP-activated protein kinase. Diabetologia 2012; 55: Zhang Y, Han P, Wu N, He B, Lu Y, Li S, Liu Y, Zhao S, Liu L, Li Y. Amelioration of lipid abnormalities by alpha-lipoic acid through antioxidative and antiinflammatory effects. Obesity (Silver Spring) 2011; 19: Napolitano A, Miller S, Nicholls AW, Baker D, Van Horn S, Thomas E, Rajpal D, Spivak A, Brown JR, Nunez DJ. Novel gut-based pharmacology of metformin in patients with type 2 diabetes mellitus. PLoS One 2014; 9:e Alfa-lipoična kiselina smanjuje tjelesnu masu i regulira koncentraciju triglicerida u gojaznih pacijenata sa šećernom bolešću Azra Okanović 1, Besim Prnjavorac 2,3, Edin Jusufović 4,5, Rifat Sejdinović 2,6 1 Dom zdravlja Tešanj, 2 Odjeljenje za interne i plućne bolesti Opće bolnice Tešanj, 3 Farmacijski fakultet Univerziteta u Sarajevu, 4 Poliklinika za plućne bolesti Zdravstveno-nastavne ustanove Dr. Mustafa Šehović Tuzla, 5 Medicinski fakultet Univerziteta u Tuzli, 6 Univerzitet u Zenici; Bosna i Hercegovina SAŽETAK Cilj Utvrditi utjecaj alfa-liponske kiseline na smanjenje tjelesne mase i regulaciju koncentracije ukupnog holesterola, triglicerida i glukoze u gojaznih osoba sa šećernom bolešću tipa 2. Metode Prospektivno istraživanje uključilo je dvije grupe gojaznih osoba sa šećernom bolešću i znakovima periferne polineuropatije: ispitivana grupa (30 pacijenata, odnosno 15 žena i 15 muškaraca) i kontrolna grupa (30 pacijenata, odnosno 12 žena i 18 muškaraca). Svi su bili tretirani metforminom ( mg/dan). Ispitanici u ispitivanoj grupi bili su dodatno tretirani alfa-liponskom kiselinom, 600 mg/dan tokom 20 sedmica. Indeks tjelesne mase i koncentracije ukupnog holesterola, triglicerida i glukoze u serumu upoređivani su prije i poslije tretmana. Rezultati Ispitanici tretirani alfa-liponskom kiselinom, u dozi od 600 mg/dan, imali su značajniji gubitak indeksa tjelesne mase, kao i triglicerida u poređenju s kontrolnom grupom. Nije bilo značajne razlike u koncentraciji ukupnog holesterola i glukoze u serumu između grupa. Zaključak Tretman alfa-liponskom kiselinom, u dozi od 600 mg/dan, utječe na gubitak indeksa tjelesne mase i dovodi do snižavanja koncentracije triglicerida u gojaznih osoba sa šećernom bolešću tipa 2. Alfa-liponska kiselina treba biti uzeta u obzir kao važna dodatna terapija u gojaznih bolesnika sa šećernom bolešću tipa 2. Ključne riječi: indeks tjelesne mase, serumska glukoza, lipidni status 127

26 ORIGINAL ARTICLE Positive correlation between uric acid and C-reactive protein serum level in healthy individuals and patients with acute coronary syndrome Emina Spahić 1, Sabaheta Hasić 2, Emina Kiseljaković 2, Halima Resić 3, Mehmed Kulić 4 1 Primary Health Center Zenica, 2 Department of Medical Biochemistry, Faculty of Medicine University of Sarajevo, 3 Clinic for Hemodialysis, Clinical Center University of Sarajevo, 4 Cardiology Clinic, Clinical Centre University of Sarajevo; Bosnia and Herzegovina ABSTRACT Aim To assess serum levels and correlation between uric acid (UA) and C-reactive protein (CRP) in acute coronary syndrome (ACS) and apparently healthy individuals. Methods The cross-sectional study included 116 examinees of age 44 to 83 years, distributed in two groups: 80 ACS patients including 40 with acute myocardial infarction (AMI), and 40 with unstable angina pectoris (UAP), and 36 apparently healthy (control group) individuals. Patients with ACS were hospitalized at the Cardiology Clinic, Clinical Centre Sarajevo in the period October- December Laboratory analyses were conducted by standard methods. The accepted statistical significance level was p<0.05. Corresponding author: Emina Spahić Primary Health Center Zenica Fra Ivana Jukića 2, Zenica, Bosnia and Herzegovina Phone ; Fax; ; spahica@hotmail.com Original submission: 14 May 2015; Revised submission: 20 June 2015; Accepted: 29 June doi: / Results Serum levels of CRP and UA were higher in patients with ACS as compared to control group (p<0.01). The median serum UA was insignificantly lower, and CRP was significantly higher in patients with AMI compared to UAP (p=0.118 and p=0.001, respectively). CRP and UA correlated positively in both ACS and control groups (rho=0.246; p=0.028 and rho=0.374; p=0.027). A positive correlation between serum CRP and UA was noted in patients with AMI, but negative in patients with UAP (p>0.05). Conclusion The correlation between CRP and UA in the patients with ACS indicates the association of oxidative stress and inflammation intensity in damaged cardiomyocytes. Correlation between UA and CRP in apparently healthy individuals indicates a possible role of UA as a marker of low-grade inflammation and its potential in risk assessment in cardiovascular diseases. Key words: angina, unstable, inflammation, myocardial infarction, oxidative stress Med Glas (Zenica) 2015; 12(2):

27 Spahić et al. Uric acid and C - reactive protein correlation INTRODUCTION Inflammation plays a significant role in all stages of atherosclerosis from initiation through progression, but also in pathogenesis of acute cardiovascular event (1). Acute inflammatory response is a major characteristic of cardiovascular occlusive event. Significant increase of serum C-reactive protein (CRP) occurs in inflammatory processes of different etiology, as a part of or independently of cardiac etiology (1,2). In healthy individuals, CRP circulates in very low concentrations (2), and individuals who are at the risk of development of cardiovascular diseases show systemic inflammatory response registered by increased serum CRP level. Apart from being a marker of inflammation, CRP contributes to development of atherosclerotic disease, and it is considered as the most powerful predictor of myocardial infarction and stroke (3). The elevated uric acid (UA) levels are related to arterial hypertension, systemic inflammation, and cardiovascular diseases through endothelial dysfunction and pathological remodeling of blood vessels, but physiological UA levels act as a powerful antioxidant (4). One of the mechanisms of elevated level of serum UA that contributes to systemic inflammation is induction of CRP expression in endothelial and smooth muscle cells of blood vessels, which facilitates its increased synthesis and elevated level in circulation (5). The CRP induces proinflammatory cytokine release and forms terminal complement complexes in the intima of the early atherosclerotic lesion which contribute to plaque instability (6). The study objective was to assess a correlation between the serum UA and CRP levels in apparently healthy individuals and acute coronary syndrome (ACS) patients. EXAMINEES AND METHODS The cross-sectional study included 116 examinees of both genders (59 males and 57 females), aged 44 to 83. Examinees were distributed into two groups: acute coronary syndrome group (ACS), consisted of 40 acute myocardial infarction patients (AMI) and 40 unstable angina pectoris (UAP), and group consisted of apparently healthy individuals as control group (CG). The patients with ACS were hospitalized at the Intensive Care Unit of Cardiology Clinic, Clinical Centre of Sarajevo in the period October- December Diagnosis of acute coronary syndrome was established on the basis of electrocardiogram changes (ECG), clinical symptoms, and elevated levels of serum cardiac troponin (ctni). The control group included apparently healthy individuals, who were without signs of diseases and who underwent a routine laboratory check. Results of biochemical analysis obtained within 48 hours of hospital admission were collected from patients medical histories. Stable angina pectoris patients and those with malignant, liver and kidneys diseases, acute or chronic systematic inflammatory diseases, infectious or septic states, patients treated with allopurinol and chronic alcohol consumption were excluded from the study. The data of control group are drawn from a database from individuals whose laboratory values were within physiological range during routine check.. Biochemical analyses were conducted at the Clinic of Chemistry and Biochemistry, Clinical Centre of Sarajevo. The serum ctni was measured by AxSYM Troponin-I ADV Immunoassay (reference range ng/ml) using AxSym analyzer (Abbott Laboratories, Abbott Park, IL, USA). Immunoturbidimetric method of CRP (reference range 0-5 mg/l) and spectrophotometric method of uric acid ( μmol/l) were used for analysis on Dimension Xpand Plus (Siemens, Munich, Germany). The study was performed according to the principles outlined in the Declaration of Helsinki. This investigation was approved by Faculty of Medicine, University of Sarajevo, Bosnia and Herzegovina. Normality of distribution of variables analyzed by Shapiro-Wilk test was not satisfied, so the numeric variables were presented by median with interquartile range (25-75 percentiles). The difference between groups was analyzed by Mann- Withney U, non-parametric test. The degree of correlation was examined by the test according to Spearman. Levels p<0.05 were considered as statistically significant. 129

28 Medicinski Glasnik, Volume 12, Number 2, August 2015 RESULTS The patients with acute coronary syndrome were slightly older in the comparison to the control group (p>0.05) (Table 1). Table 1. Demographic characteristics of patients with acute coronary syndrome and controls Variables CG (n=36) ACS (n=80) AMI (n=40) UAP (n=40) Age (years) 64 (44-81)* 67 (50-83) 66 (53-81) 67 (50-83) No (%) of males 17 (47.2) 40 (50) 20 (50) 20 (50) *NS (not significant) compared to ACS patients (p>0.05) CG, control group; ACS, acute coronary syndrome; AMI, acute myocardial infarction; UAP, unstable angina pectoris; Median level of serum ctni in the ACS patients was 0.33 ( ) ng/ml and AMI patients had significantly higher ctni level in the comparison to UAP patients (p<0.01). Levels of CRP and UA were significantly higher in the ACS compared to the control group (p<0.01). Levels of CRP in AMI patients were higher in comparison to UAP group (p=0.001), but UA was lower compared to UAP (p=0.118) (Table 2). Table 2. Blood levels of cardiac troponin I, C-reactive protein and uric acid in acute coronary syndrome patients and controls Variables Patient s group ctni (ng/ml) CRP (mg/l) UA (µmol/l) ACS ( ) ( )* ( )* CG / AMI UAP ( ) 0.06 ( ) 1.90 ( ) 47.5O ( ) ( ) 263 ( ) ( ) 379 ( ) *p<0.01 between ACS and control group; p<0.01 between AMI and UAP groups; no significant difference between and AMI and UAP groups; ACS, acute coronary syndrome; CG, control group; AMI, acute myocardial infarction; UAP, unstable angina pectoris; ctni, cardiac troponin I; CRP, C reactive protein; UA, serum uric acid A moderate positive relationship between serum CRP and UA levels (rho=0.374; p=0.027) was observed in the CG patients (Table 3), while weaker relationship between the two observed parameters was noticed in ACS group (rho=0.246, p=0.028). Although the association between CRP and UA was positive in AMI and negative in UAP group, both of them were no significant (p>0.05). Table 3. Spearman s correlation of serum C-reactive protein and uric acid in patients with acute coronary syndrome and in control group Patient groups Variables CG ACS AMI UAP rho CRP / UA p 0.027* 0.028* *p<0.05; CRP, C-reactive protein; UA, serum uric acid; CG, control group; ACS, acute coronary syndrome; AMI, acute myocardial infarction; UAP, unstable angina pectoris; rho-spearman correlation coefficient; DISCUSSION The development of atherosclerosis depends on the balance between proinflammatory stimuli, anti-inflammatory and anti-oxidative defense mechanisms. The range of CRP values in blood of healthy volunteers indicates a low grade inflammation (CRP > 3 mg/l). According to American Heart Association, this category of apparently healthy individuals is at the risk of development of cardiovascular diseases (6,7). Yamada et al. have found that in 94% of the apparently healthy individuals CRP was lower than 2 mg/l with median of 0.12 mg/l (8). In Koenig et al. study, % of the individuals had CRP lower than 2 mg/l, which is considered as a level for detection of active inflammation, infection, or tissue damage (9). Investigation of serum UA concentration and its role and importance as an independent risk factor for the development of cardiovascular diseases is complicated by the fact that elevated levels of UA combine with other factors to increase the cardiovascular risk (10). The physiological range of uric acid concentration has been noted in healthy individuals included in the present study. As one of the most powerful antioxidants, uric acid eliminates free radicals from the body, and serum UA elevation could represent a compensatory mechanism against free radicals (10). Uric acid functions in early stages of atherosclerosis as an antioxidant, but it becomes prooxidant in the cell due to the increased rate of xanthine oxidase activity (11). In prospective cohort study which included middle- aged Finish men without cardiovascular diseases, cancer or diabetes, serum UA levels in the lower third of reference range were associated with greater risk of cardiovascular death than those with concentrations of UA in upper third. (12). Consistent with Jalal at al. study (13), we observed a positive association between CRP and UA in the control group. De Carvalho Vidigal study suggested that uric acid is suitable biochemical indicator for detecting changes in hs-crp and can also predict higher C-reactive protein levels in apparently healthy men improving the assessment of cardiovascular risk (14). Serum levels of CRP in the present study were significantly higher in ACS subjects compared to the controls, which is in accordance with Baruah et al. and Kushner et al. studies (15,16). Myocar- 130

29 Spahić et al. Uric acid and C - reactive protein correlation dial necrosis leads to inflammatory response, cytokines activation and consequential increase of CRP synthesis. Binding of CRP to necrotic myocardial cells and consecutive complement activation is considered responsible for a further myocardial necrosis expansion (17). Higher level of serum CRP concentration in the patients with AMI indicates greater myocardial inflammatory response as the consequence of more severe myocardial lesion in AMI than in UAP patients (18). In Munir et al. study, significant increase of serum CRP within 12 to 24 hours has been shown in patients with UAP, myocardial infarction without elevation of ST-segment (NSTEMI), and myocardial infarction with ST elevation (STEMI) (19). Besides the CRP localization in atherosclerotic lesions, it localizes also in ischemic myocardium and promotes complement activation (20). Results of this study have shown that levels of UA in ACS group were statistically significantly higher in comparison with the control group, and insignificantly lower in the AMI group compared to UAP (p=0.118). The similar results were published in Gur et al. study who found increased UA values in AMI and UAP patients compared with controls, but uric acid level was not in relation to the severity of coronary artery disease (21). Higher levels of uric acid in- REFERENCES 1. Shrivastava AK, Singh H V, Raizada A, Singh SK. C-reactive protein, inflammation and coronary heart disease. The Egyptian Heart Journal 2015; 67: Matunović R, Stojanović A, Mijailović Z, Rađen G. Importance of determining biomarkers of myocardial necrosis in acute coronary syndrome. Vojnosanit Pregl 2005; 62(Suppl 5): Ridker PM. High-sensitivity C-reactive protein: Potential adjunct for global risk assessment in the primary prevention of cardiovascular disease. Circulation 2001; 103: Rodrigo R, Libuy M, Feliú F, Hasson D. Oxidative stress-related biomarkers in essential hypertension and ischemia-reperfusion myocardial damage. Dis Markers 2013; 35(Suppl 6): Car S. Serum concentration of uric acid and the outcome of acute coronary syndrome. Medical Faculty, University of Zagreb; 2012; Ph. D. thesis. 6. Silvaa D, Lacerda AP. High-sensitivity C-reactive protein as a biomarker of risk in coronary artery disease. Rev Port Cardiol. 2012; 31(Suppl 11): Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO III, Criqui M. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for health care professionals from the Centers for Disease Condicate greater intensity of oxidative stress in ACS than in control group. Under local ischemia condition and increased synthesis of oxygen radicals, UA becomes prooxidant (22-24). We have notified that levels of CRP and UA positively correlated in ACS patients. In Baruah et al. study, the similar kinetic of CRP and UA have been registered in post-infarction period. Both markers peak on the 3 rd day, and then return to baseline level (15). In the present study a positive correlation between CRP and UA in the serum was established in AMI, and a negative correlation in UAP group, but it was not statistically significant. We consider that a positive correlation between uric acid and C-reactive protein in serum of healthy individuals indicates the importance of serum levels of uric acid in monitoring the intensity of low-grade inflammation. The correlation between the two observed parameters in acute coronary syndrome indicates correlation between intensity of oxidative stress and inflammation in myocardial tissue. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. trol and Prevention and the American Heart Association. Circulation 2003; 107(Suppl 3): Yamada S, Gotoh T, Nakashima Y, Kayaba K, Ishikawa S, Nago N. Distribution of serum C-reactive Protein and Its Association with Atherosclerotic Risk Factors in a Japanese Population. Am J Epidemiol. 2001; 153(Suppl 12): Koenig W, Sund M, Frohlich M, Fischer HG, Löwel H, Döring A. C-Reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men. Augsburg Cohort Study, 1984 to1992. Circulation 1999; 99: Nieto F, Iribarren C, Gross M, Comstock GW, Cutler RG. Uric acid and serum antioxidant capacity: a reaction to atherosclerosis. Atherosclerosis 2000; 148(Suppl 1): Hayden MR, Tyagi SC. Uric acid: a new look at an old risk marker for cardiovascular disease, metabolic syndrome, and type 2 diabetes mellitus: the urate redox shuttle. Nutr Metab. 2004; 19(Suppl 1): Niskanen LK, Laaksonen DE, Nyyssonen K, Alfthan G, Lakka HM, Lakka TA. Uric acid level as a risk factor for cardiovascular and all-cause mortality in middle-aged men: a prospective cohort study. Arch Intern Med. 2004; 165(Suppl 9):

30 Medicinski Glasnik, Volume 12, Number 2, August Jalal D, Jablonski K, McFann K, Choncholand M, Seals D. Vascular Endothelial Function Is Not Related to Serum Uric Acid in Healthy Adults. Am J Hypertens. 2012; 25(Suppl 4): Vidigal F, Rosado L, Rosado GP, Ribeiro C. The high UA levels with increase of CRP and inflammation in the body can contribute to progression of atherosclerotic disease. C. Franceschini Sdo C. Nutr Hosp. 2014; 29(Suppl 4): Baruah M, Nath C, Chaudhury B, Devi R, Ivvala AS. A study of serum uric acid and C-reactive protein in acute myocardial infarction. IJBMSP 2012; 2(Suppl 1): Kushner I, Broder M, Karp D. Control of the acute phase response. Serum C-reactive protein kinetics after acute myocardial infarction. J Clin Invest 1998; 61(Suppl 2): Swiatkiewicz I, Kozinski M, Magielski P, Fabiszak T, Sukiennik A, Navarese EP, Odrowaz-Sypniewska G, Kubica J. Value of C-reactive protein in predicting left ventricular remodelling in patients with a first ST-segment elevation myocardial infarction. Mediators Inflamm 2012; 2012: Yildiz HT. High Sensitive C - reactive protein levels in patients with acute coronary syndrome. The Iraqi Postgraduate Medical Journal 2013: Munir TA, Afzal MN, Ahmed R. C-reactive protein and acute coronary syndrome: correlation with traditional risk factors, diagnostic cardiac biomarkers, and ejection fraction. RMJ. 2009; 34(Suppl 2): Lagrand WK,Visser CA, Hermens WT, Niessen HWM,Verheught FWA, Wolbink GJ, et al. C-reactive protein as a cardiovascular risk factor; more than an epiphenomenon. Circulation 1999; Gur M, Yilmaz R, Demirbag R, Aksoy N. Relation of serum uric acid levels with the presence and severity of angiographic coronary artery disease. Angiology 2008; 59(Suppl 2): Strazzullo P, Puig JG. Uric acid and oxidative stress: relative impact on cardiovascular risk? Nutr Metab Cardiovasc Dis 2007; 17(Suppl 6): Sautin YY, Johnson YR. Uric Acid: The oxidant-antioxidant paradox. Nucleosides Nucleotides Nucleic Acids 2008; 27(Suppl 6): Ndrepepa G, Braun S, Haase HU, Schulz S, Ranftl S, Hadamitzky M, Mehilli J, Schömig A, Kastrati A. Prognostic value of uric acid in patients with acute coronary syndromes. Am J Cardiol 2012; 109(Suppl 9): Pozitivna korelacija između mokraćne kiseline i C-reaktivnog proteina u serumu zdravih osoba i pacijenata s akutnim koronarnim sindromom Emina Spahić 1, Sabaheta Hasić 2, Emina Kiseljaković 2, Halima Resić 3, Mehmed Kulić 4 1 Dom zdravlja Zenica, 2 Katedra za medicinsku biohemiju, Medicinski fakultet, Univerzitet u Sarajevu, 3 Klinika za hemodijalizu, 4 Klinika za srce i reumatizam; Klinički centar Univerziteta u Sarajevu; Bosna i Hercegovina SAŽETAK Cilj Ispitati serumske vrijednosti i odnos između mokraćne kiseline (MK) i C-reaktivnog proteina (CRP) kod naizgled zdravih osoba i kod pacijenata s akutnim koronarnim sindromom (AKS). Metode U presječnu studiju bilo je uključeno 116 ispitanika, starosne dobi između 44 i 83 godine, distribuiranih u dvije grupe: 80 ispitanika s AKS-om i to 40 s akutnim infarktom miokarda (AIM) i 40 s nestabilnom anginom pektoris (NAP), te 36 naizgled zdravih ispitanika (kontrolna grupa, KG). Ispitanici s AKS-om bili su hospitalizirani na Klinici za bolesti srca i reumatizam Univerzitetskog kliničkog centra u Sarajevu u periodu od oktobra do decembra godine. Laboratorijske analize su urađene korištenjem standardnih metoda. Prihvaćeni nivo statističke značajnosti iznosio je p<0.05. Rezultati Vrijednosti CRP-a i MK-a u serumu bile su više u ispitanika s AKS-om u odnosu na KG (p<0.01). Vrijednost MK-a u serumu nije bila značajno niža, dok je vrijednost CRP-a bila značajno viša kod pacijenata s AIM-om u poređenju s pacijenatima s nestabilnom anginom pektoris (p=0.118; p=0.001). Pozitivna, značajna korelacija je utvrđena između serumskih vrijednosti CRP-a i MK-a i kod ispitanika s AKS-om i kod KG-a. (rho=0.246; p=0.028 i rho=0.374; p=0.027). Utvrđen je pozitivan odnos CRP-a i MK-a u serumu ispitanika s AIM-om, a negativan u NAP-u, ali nije bio statistički značajan (p>0.05). Zaključak Odnos CRP-a i MK-e u serumu oboljelih od AKS-a ukazuje na povezanost intenziteta oksidativnog stresa s intenzitetom inflamacije zahvaćenog dijela kardiomiocita. Povezanost vrijednosti MK-a i CRP-a u serumu naizgled zdravih ispitanika ukazuje na moguću ulogu MK-a kao markera intenziteta hronične inflamacije niskog stepena i ima potencijal u procjeni rizika za razvoj kardiovaskularnih bolesti. 132

31 ORIGINAL ARTICLE Role of echocardiography in diagnosis and management of complete papillary muscle rupture caused by myocardial infarction Josip Vincelj 1,2,3, Stanko Biočić 1, Mario Udovičić 1, Mario Sičaja 1, Sandra Jakšić Jurinjak 1 1 Institute of Cardiovascular Diseases, Dubrava University Hospital, Zagreb, 2 University of Applied Health Studies; Zagreb, 3 School of Medicine, University J. J. Strossmayer, Osijek; Croatia ABSTRACT Aim To evaluate the usefulness of echocardiography in the diagnosis of complete rupture of papillary muscle. Methods Transthoracic (TTE) and transesophageal echocardiography (TEE) was performed with the ATL 3000 HDI Ultrasound Inc (Bothell, WA, USA) with a 2.5 MHz transducer and 5-7 MHz multiplane phased array transducer. We are reporting about two patients (a 45 and a 51-year old male) with complete ruptures of papillary muscle following acute myocardial infarction (AMI). Corresponding author: Josip Vincelj Dubrava University Hospital, Institute of Cardiovascular Diseases Av. G. Šuška 6, Zagreb, Croatia Phone: ; Fax: ; jvincelj@kbd.hr Results Both patients were previously treated with fibrinolysis in their local hospitals, 400 and 300 km, respectively, away from our hospital. Massive mitral regurgitation developed in both followed by rapid deterioration of hemodynamic state and severe heart failure, because of which both were transferred by helicopter to the Coronary Care Unit of our clinic. The diagnosis of complete papillary muscle rupture was confirmed in both patients by TTE and TEE. Due to the significant deterioration in their hemodynamic state, vasoactive drugs and intra-aortic balloon pump support were applied. Both patients then underwent mitral valve replacement, accompanied by concomitant coronary artery bypass grafting in one case. Conclusion Transesophageal echocardiography is a more accurate and rapid diagnostic method in patients with mechanical complications of AMI than TTE. Original submission: 19 November 2014; Revised submission: 15 January 2015; Accepted: 27 April doi: / Keywords: mitral valve, replacement, coronary artery, bypass grafting Med Glas (Zenica) 2015; 12(2):

32 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION Papillary muscle rupture is a rare, but often fatal mechanical complication of acute myocardial infarction (AMI); it occurs in 1% to 5% of patients with AMI (1). Echocardiography is the imaging technique of choice for detecting mechanical complications of AMI including myocardial free wall rupture, acute ventricular septal defect and mitral regurgitation secondary to papillary muscle rupture or ischemia (2,3). Transesophageal echocardiography is more sensitive than TTE, and useful for providing more detailed and/or unique anatomic information in patients with papillary muscle rupture than TTE (3,4). Minami et al (5) have reported on six patients (from 1986 to 2002) with posterior (n=4) and anterior (n=2) papillary muscle rupture; all patients underwent mitral valve replacement, concomitant coronary artery bypass grafting (CABG) was performed in five of six patients, and the perioperative mortality rate was 33%. Tavakoli et al (6) reviewed 21 consecutive patients (from 1988 to 1998) with a perioperative mortality of 19%. Transthoracic (TTE) and/or transesophageal echocardiography (TEE) established the diagnosis of papillary muscle rupture in 14 patients, and in others the diagnosis was suspected on the basis of the presence of flail mitral leaflets (3). Emergency surgery, even as a salvage procedure for acute postinfarction mitral papillary muscle rupture is justified (6). The aim of this study was to evaluate the usefulness of echocardiography in the diagnosis of complete rupture of papillary muscle, based on a presentation of two male patients with massive mitral regurgitation and severe heart failure due to complete mitral papillary muscle rupture following acute myocardial infarction. PATIENTS AND METHODS Dubrava University Hospital provides acute care for about inhabitants from the east Zagreb area including Sesvete, DugoSelo, Vrbovec and Sv. Ivan Zelina. Our hospital is also one of the primary percutaneous coronary intervention(pci) centers included in primary-pci network of Croatia which provides care for patients with acute coronary syndrome for Međimurje County, Koprivnica - Križevci County, Bjelovar - Bilogora County and east part of the Zagreb County. About 750 patients with acute coronary syndrome are admitted to our hospital per year. Diagnosis of AMI included clinical symptoms, electrocardiogram, cardiac biomarkers (troponin I, normal limit is 0.04 ug/ml), echocardiography and coronary angiography. Definite diagnosis of AMI requires cardiac biomarker troponin with at least one value above the 99 th percentile of the upper reference limit and with at least one of the following: symptoms of ischemia, new or presumably new significant ST-T changes or new left bundle branch block (LBBB), development of pathologic Q waves in the ECG, imaging evidence of new loss of viable myocardium and identification of an intracoronary thrombus by angiography or autopsy (2). The diagnosis of heart failure was based on typical symptoms, typical signs and relevant structural heart disease. Patients symptomatic limitation is graded using the New York Heart Association (NYHA) functional classification (class I - IV). This classification assigns patients to one of four class depending on the degree of effort needed to elicit symptoms of angina, fatigue, dyspnea or palpitation. Patients with NYHA IV class presenting with symptoms at rest and unable to carry out any physical activity without discomfort. Transthoracic (TTE) and transesophageal echocardiography (TEE) was performed with the ATL 3000 HDI Ultrasound Inc (Bothell, WA, USA) with a 2.5 MHz transducer and 5-7 MHz multiplane phased array transducer.the presence and grade of mitral regurgitation (MR) were screened by color-flow imaging with the MR jet area to left atrium area ratio; a ratio of >20%, 20-40% and >40% represents mild, moderate and severe MR, respectively. In patients with more than trace regurgitation, the regurgitant orifice area was calculated by the proximal isovelocity surface area (PISA) method and the degree of MR was graded as mild (regurgitant orifice area <0.2cm 2 ), moderate (regurgitant orifice area 0.2 to 0.4 cm 2 ), or severe (regurgitant orifice area >0.4cm 2 ). Classically a vena contracta width <3mm indicates mild MR, whereas a vena contracta width 7 mm indicates severe MR (7). 134

33 Vincelj et al. Echocardiography and papillary muscle rupture RESULTS Case 1 A 45-year-old man was first admitted to a local hospital for chest pain, faintness, dyspnea and waning exercise tolerance. This hospital is approximately 400 km away from our clinic. Treatment consisted of fibrinolytic therapy with streptokinase, along with other standard therapeutic regimens. Transthoracic echocardiography showed mitral regurgitation (MR) grade 3, and in spite of aggressive therapy, the patient began developing severe hemodynamic instability. After a week-long clinical deterioration he was transferred to the coronary care unit (CCU) of our clinic. He presented with clinical signs of severe heart failure,nyhaclass IV, while physical examination revealed bilateral basal pulmonary rales without jugular venous distension. On careful auscultation soft heart sounds and a holosystolic heart murmur with a point of maximum intensity at the apex grade 4/6 were detected. Blood pressure was 140/80 mm Hg, accompanied by tachycardia 133 beats/min. Electrocardiogram (ECG) revealed sinus tachycardia of 133 beats/min and Q-waves of the left ventricular inferoposterior wall. Chest radiography disclosed cardiac enlargement on the left side and pulmonary plethora. In laboratory findings there was increased aspartate aminotransferase 55 U/L, alanine aminotransferase 51 U/L, lactate dehydrogenase 765 U/L, uric acid 698 U/L, total plasma cholesterol 6.09 mmol/l, LDL-cholesterol 4.36 mmol/l, and fibrinogen 7.8 g/l. Transthoracic echocardiography and TEE discovered mitral regurgitation grade 4. There was a mobile solid mass of 1.6x0.6 cm in size on the posterior mitral leaflet, of the same echo-dense structure as the myocardium and suggestive of complete rupture of the head of the posteromedial papillary muscle. The ruptured head of the papillary muscle was detected in the left ventricle during diastole accompanied by its systolic displacement displaced to the left atrium during systole (Figure 1). Echocardiography showed hypokinesis of the left ventricular inferoposterior wall with normal ejection fraction (LVEF=65%). Ventriculography confirmed the diagnosis of severe MR with normal left ventricular function. Coronary angiography detected significant stenosis of the circumflex, the first obtuse marginal and of the right coronary artery, as well as occlusion Figure 1. Transesophageal echocardiogram showing complete rupture of the head of posteromedial papillary muscle prolapsing into the left atrium (arrow) (Vincelj J, 2004) LA, left atrium; LV, left ventricle; AO, aorta. of the second obtuse marginal artery. Due to the significant fluctuations in the hemodynamic state of the patient, vasoactive drugs and the intra-aortic balloon pump (IABP) were applied. Ten days after AMI, the patient underwent mitral valve replacement with a Carbomedics mechanical valve and concomitant coronary artery bypass grafting of the circumflex artery. During surgery an intraoperative TEE also confirmed the diagnosis of complete posteromedial papillary muscle rupture (Figure 2). The patient was weaned from cardiopulmonary bypass without hemodynamic disturbances and 24 hours later he was also successfully weaned from both mechanical ventilation and IABP. Transthoracic echocardiography performed in the early postoperative course diclosed normal function of mechanical mitral valve and normal left ventricular function. Histopathological analysis found ischemic necrosis of the posteromedial papillary muscle and fibroelastic thickness of the endocardium. A part of the mitral leaflet showed fibroelastic thickness to some extent. Microbiology results showed a sterile papillary muscle and leaflet. Figure 2. Specimen of the resected ruptured posteromedial papillary muscle (Vincelj J, 2004) 135

34 Medicinski Glasnik, Volume 12, Number 2, August 2015 Case 2 A 51-year-old man was treated for acute ST-segment elevation myocardial infarction (STEMI) of inferior localization in his local hospital, 300 km away from our clinic. Treatment consisted of fibrinolytic therapy with streptokinase, along with other standard therapy. During the first week of hospitalization, acute heart failure emerged and the patient started exhibiting signs of rapid hemodynamic deterioration. On day 12, he was transferred to the CCU of our hospital. He presented with clinical signs of severe heart failure, NYHA class IV. Physical examination showed bilateral basal pulmonary rales without jugular venous distension. Careful auscultation revealed calm heart sounds and holosystolic heart murmur grade 3/6. Blood pressure was 105/65 mm Hg, accompanied by sinus tachycardia 120 beats/min. ECG revealed sinus tachycardia of 120 beats/min and Q-waves of the left ventricular inferoposterior wall. Chest radiography disclosed cardiac enlargement on the left side and pulmonary plethora. His routine laboratorytest revealed increased creatine kinase 254 U/L, aspartate aminotransferase 36 U/L, alanine aminotransferase 74 U/L, lactate dehydrogenase 775 U/L, uric acid 452 U/L, LDL-cholesterol 3.74 mmol/l, and fibrinogen 10.4 g/l. Transesophageal echocardiography exposed a completely ruptured head of the anterolateral papillary muscle prolapsing into the left atrium with massive MR. There was a display of chaotic movement of mitral valve. Excessive amount of pericardial effusion (20 mm) was seen, along with a collapse of the right atrium in telediastole, as sign of impending tamponade (Figure 3). Coronary angiography detected Figure 3. Transesophageal echocardiogram showing complete rupture of the head of anterolateral papillary muscle (P) prolapsing into the left atrium (Vincelj J, 2004) LA, left atrium; LV, left ventricle; RA, right atrium; PE, pericardial effusion. a subtotal stenosis of first obtuse marginal artery along with a non-significant ostial stenosis of hypoplastic right coronary artery. The patient was referred for urgent cardiac surgery. Being unsuitable for surgical revascularization, he only underwent mitral valve replacement with a Carbomedics mechanical valve 24 days after AMI.An intraoperative TEE was also performed. Postoperative course of the patient (with known prior medical history of obstructive lung disease) was complicated by a respiratory infection and two episodes of bronchospasm, which were both successfully treated. Rapid postoperative improvement of cardiac function and regression of congestion in pulmonary circulation ensued, and echocardiography performed during postoperative course revealed normal function of the mechanical mitral valve and no impairment of left ventricular systolic function. Histopathological analysis showed ischemic necrosis of the anterolateral papillary muscle. The patient was discharged, fully recovered, 14 days after surgery. Both patients fully recovered and were discharged from hospital 8 and 14 days, respectively, after surgery. DISCUSSION Papillary muscle rupture is a rare but often fatal mechanical complication of AMI with hemodynamic instability (4,8). The involvement of the posteromedial papillary muscle is 6-12 times more common than that of the anterolateral (4,8). Indeed, posteromedial papillary muscle vascularization is provided only by the interventricular posterior coronary artery originating even from the right coronary artery or from the circumflex coronary artery, and it may aggravate infarction heralded by occlusion in such vessels (8). Echocardiography is the imaging technique of choice for detecting mechanical complications of AMI including myocardial free wall rupture, acute ventricular septal defect, and mitral regurgitation secondary to papillary muscle rupture or ischemia (2). Transthoracic echocardiography is able to identify a papillary muscle rupture with a diagnostic sensitivity of 65-85%. Transesophageal echocardiography is more sensitive than TTE (4,9). Recently, TEE has been reported as a valuable adjunct to conventional echocardiography, by providing more detailed and/or unique anatomic information in patients 136

35 Vincelj et al. Echocardiography and papillary muscle rupture with papillary muscle rupture involving either mitral or tricuspid valve apparatus, to either ischemic or traumatic damage.when the head of the papillary muscle is ruptured, it can often be imaged prolapsing into the left atrium. In up to 30% of patients, however, the ruptured head may not prolapse into the left atrium. In this case the diagnosis is made by noting chaotic movement of the ruptured head in the left ventricle (10). Complete papillary muscle rupture may cause acute MR with pulmonary edema or cardiogenic shock (8). These events depend on the infarction severity and papillary muscle morphology. There are morphology differences in papillary muscle attachment to the left ventricular wall. The main part of the posteromedial papillary muscle may be attached to the left ventricular wall with one, two or three heads, whereby the papillary muscle heads maybe attached separately or jointly (3). Furthermore, the cordae tendineae of one papillary muscle may be attached to both mitral leaflets. In papillary muscle rupture, the grade of mitral regurgitation depends on rupture severity (complete or incomplete) and number of papillary muscle heads. The regurgitation jet will be smaller if the papillary muscle has two or three heads, only one of them being involved by rupture. Severe mitral regurgitation develops upon complete rupture of a papillary muscle which has only one or two heads, or complete rupture of all three muscle heads, depending on differences in the muscle morphology (11). In our patients, the posteromedial and anterolateral papillary muscle was attached to the muscle wall with one head, while chordae tendineae were attached to the anterolateral leaflet, thus additionally contributing to MR. In this case mitral valve repair was impossible for two reasons. First, myocardial necrosis caused complete posteromedial papillary muscle rupture; and second, chordae tendineae of the posteromedial papillary muscle were in part attached to the anterolateral leaflet (A2 scallop) thus also participating in severe MR. REFERENCES 1. Wei JY, Hutchins GM, Bulkley BH.Papillary muscle rupture in fatal acute myocardial infarction: a potentially treatable form of cardiogenic shock. Ann Intern Med 1979;90: Thygesen K, Alpert JS, White HD. Joint ESC/ ACCF/AHA/WHF Task Force for the Redefinition of Myocardial infarction. Universal definition of myocardial infarction. Eur Heart J 2007; 28: Our patients have some distinctive features related to prior published data. Distinctive features of our cases are that both patients were being treated for AMI in hospitals 400 and 300 km away from our hospital. Initially, in both cases the diagnosis of papillary muscle rupture was suspected, and as rapid hemodynamic destabilization became evident, both patients were transported, as safely and as fast as possible. Optimal cooperation and coordination were crucial for successful outcome. This relates to interregional and regional cooperation, but also to collaborative work between cardiologists and cardiac surgeons, so called hybrid strategy, which included early reperfusion, when possible, and subsequent mitral valve surgery (12). Fibrinolytic therapy has positive effect on mechanical complications in AMI.The study of Gueret et al. demonstrated a dramatic reduction in the incidence of mechanical complications of AMI in the reperfusion era when compared with the non reperfusion era. Results of study documented beneficial effect of thrombolysis on some of these complications, whereas similar information after primary coronary angioplasty is scarce (13). Transesophageal echocardiography as an accurate and rapid diagnostic method is highly important in patients with papillary muscle rupture in AMI. The role of coronary angiography is mandatory before making any therapeutic decision. Surgery, which should be ensured without delay, was performed after three days of treatment, with a satisfactory result. In our patients, urgent transportation, accurate and rapid diagnostics, hemodynamic stabilization and timely surgery were all crucial for successful outcome. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. 3. Nixdorff U, Rupprecht HJ, Mohr-Kahaly S, Wittlich N, Oelert H, Schmied W, Meyer J.Transesophageal echocardiography in cardiogenic shock in acute posterior wall infarct with rupture of the papillary muscles. Z Kardiol 1994; 83:

36 Medicinski Glasnik, Volume 12, Number 2, August Lunghetti S, D Asaro MG, Guerrieri G, Zaca V, Carrera A, Fusi S, Padeletti M, Mondillo S, Favilli S. Massive mitral regurgitation secondary to acute ischemic papillary muscle rupture: the role of echocardiography. Cardiol J 2010; 17: Minami H, Mukohara N, Obo H, Yoshida M, Nakagiri K, Hanada T, Maruo A, Matsuhisa H, Morimoto N, Shida T. Papillary muscle rupture following acute myocardial infarction. Jpn J Thorac Cardiovasc Surg 2004;52: Tavakoli R, Weber A, Vogt P,Brunner HP, Pretre R, Turina M.Surgical management of acute mitral valve regurgitation due to post-infarction papillary muscle rupture. J Heart Valve Dis 2002;11: Kossaify A, Akiki V. Echocardiographic assessment of mitral valve regurgitation, pattern and prevalence, expanding clinical awareness through an institutional survey with the perspective of a quality improvement project. Clin Med Insight Cardiol 2014;8: Slater J, Brown RJ, Antonelli TA,Menon V, Bolant J, Col J, Dzavik V, Greenberg M, Menegus M, Connery C, Hochman JS.Cardiogenic shock due to cardiac free-wall rupture or tamponade after acute myocardial infarction: a report from the SHOCK Trial registry. Should we emergently revascularize occluded coronaries for cardiogenic shock?j Am CollCardiol 2000;36: Czarnecki A, Thakrar A, Fang T, Lytwyn M, Ahmadie R, Pascoe E, Jassal DS. Acute severe mitral regurgitation: consideration of papillary muscle architecture. Cardiovasc Ultrasound 2008;6: Nanda NC, Domanski MJ. Atlas of transesophageal echocardiography. Baltimore: Williams & Wilkins, Berdajs D, Lajos P, Turina M. A new classification of the mitral papillary muscle. Med SciMonit 2005;11: Jessurun GA, Erasmus ME, Wijpkema JS, Rietman GW, Tio RA. Hybrid treatment strategy for papillary muscle rupture in acute myocardial infarction. Int J Cardiol 2004;96: Gueret P, Khalife K, Jobic Y, Fillipi E, Isaaz K, Tassan-Mangina S, Baxias C, Motreff P, Meune C. Echocardiographic assessment of the incidence of mechanical complications during the early phase of myocardial infarction in the reperfusion era: a French multicentre prospective registry. Arch Cardiovasc Dis 2008;101:41-7. Uloga ehokardiografije u dijagnostici i zbrinjavanju kompletne rupture papilarnog mišića uzrokovane infarktom miokarda Josip Vincelj 1,2,3, Stanko Biočić 1, Mario Udovičić 1, Mario Sičaja 1, Sandra Jakšić Jurinjak 1 1 Zavod za bolesti srca i krvnih žila, Klinička bolnica Dubrava Zagreb, 2 Zdravstveno veleučilište; Zagreb, 3 Sveučilište J. J. Strossmayera, Osijek, Medicinski fakultet, Osijek; Hrvatska SAŽETAK Cilj Procijeniti korisnost ehokardiografije u dijagnostici kompletne rupture papilarnog mišića. Metode Za transtorakalnu (TTE) i transezofagusnu ehokardiografiju (TEE) korišten je aparat ATL 3000 HDI Ultrasound Inc. (Bothell, WA, USA), sondama od 2,5 MHz i od 5 do 7 MHz. Opisana su dva muška bolesnika u dobi 45 i 51 godine s kompletnom rupturom papilarnog mišića u akutnom infarktu miokarda (AIM). Rezultati Oba bolesnika liječena su trombolitičkom terapijom zbog AIM-a u dvije lokalne bolnice udaljene 400, odnosno 300 km. Zbog masivne mitralne regurgitacije koja je uzrokovala hemodinamsku nestabilnost i zatajivanje srca teškog stupnja bolesnici su helikopterom transportirani u koronarnu jedinicu naše Klinike. Dijagnozu kompletne rupture papilarnog mišića potvrdili smo u oba bolesnika pomoću TTE-a i TEE-a. Zbog teškog općeg stanja i hemodinamske nestabilnosti bolesnici su primali vazoaktivu terapiju i priključeni su na intraaortalnu balon-crpku. U oba bolesnika implantirana je umjetna mitralna valvula, a aortokoronarno premoštenje učinjeno je u jednog bolesnika. Zaključak Transezofagusna ehokardiografija u odnosu na TTE je točna i brza dijagnostička metoda u bolesnika s mehaničkim komplikacijama u akutnom infarktu miokarda. Ključne riječi: mitralna valvula, implantacija, koronarne arterije, premoštenje 138

37 ORIGINAL ARTICLE Impact of reperfusion therapy and infarct localization on frequency of premature ventricular beats in acute myocardial infarction Davor Horvat 1, Josip Vincelj 2,3,4 1 Internal Disease Service, General Hospital Karlovac, Karlovac, 2 Institute of Cardiovascular Diseases, Dubrava University Hospital, Zagreb, 3 University of Applied Health Studies, Zagreb, 4 School of Medicine Osijek, University J.J. Strossmayer Osijek; Croatia ABSTRACT Aim To determine the impact of infarct localization and types of reperfusion therapy on the frequency of ventricular premature beats (VPBs) in patients with acute myocardial infarction (AMI) and reduced left ventricular ejection fraction (LVEF). Methods A total of 705 patients with acute ST elevation myocardial infarction (STEMI) were divided according to the infarct localization (anteroseptal, anterolateral, inferior and posterior) and reperfusion therapy (fibrinolysis or percutaneous coronary intervention with stenting) into two groups: LVEF<45% was an experimental group and LVEF>45% was a control group. The occurrence of VPBs<10 per hour was defined as a non-significant, and the occurrence of VPBs>10 per hour defined as a significant. Corresponding author: Davor Horvat Department of Cardiology, General Hospital Karlovac Andrija Štampar 3, Karlovac, Croatia Phone: ; Fax: ; davor.horvat@ka.t-com.hr Results In patients with fibrinolysis therapy and LVEF<45% significant number of VPBs were in anteroseptal (p=0.017), anterolateral (p<0.001) and posterior AMI (p<0.001), but in patients with percutaneous coronary intervention (PCI) and LVEF<45% significant number of VPBs were only in anteroseptal AMI (p=0.001) localization. Conclusion In patients with reduced ejection fraction in AMI, treatment with PCI method has a better antiarrhythmic effect compared to fibrinolysis treatment. Key words: fibrinolysis, percutaneous coronary ventricular arrhythmias intervention, Original submission: 11 May 2015; Revised submission: 09 June 2015; Accepted: 01 July doi: / Med Glas (Zenica) 2015; 12(2):

38 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION Ventricular premature beats (VPB) represent a premature ventricular contraction (1). The association between VPBs and poor patient prognosis has been well documented (2). Ventricular premature beats in the early phase of an acute myocardial infarction (AMI) are not considered in the high mortality rate (3,4). However, after the first 48 hours, VPBs are a problem with longterm prognostic significance (5). The background might be an unsynchronized myocardial repolarization on the grounds of ischemia, lesion or myocardial necrosis (6). Patients with frequent premature ventricular contractions or certain patterns of premature ventricular contractions may be at increased risk of developing heart rhythm problems (arrhythmias) or weakening of the heart muscle (cardiomyopathy) (7-9). The difference in conduction velocity between injured and uninjured tissue can trigger re-entry or a feedback loop that is believed to be the cause of many lethal arrhythmias (10). The most serious of arrhythmias is ventricular fibrillation, an extremely fast and chaotic heart rhythm that is the leading cause of sudden cardiac death. Another life-threatening arrhythmia is ventricular tachycardia, which can cause sudden cardiac death. Rarely, when accompanied by underlying heart disease, frequent premature contractions can lead to chaotic, dangerous heart rhythms and possibly sudden cardiac death (11,12). Post-AMI VPBs, particularly if frequent (more than 10 per hour) or complex (repetitive forms, primarily nonsustained ventricular tachycardia), appear to be associated with a worse prognosis in patients with a prior AMI (10). The relationship among reduced left ventricular ejection fraction (LVEF) and the size of affected myocardial area might be also important (13). Therefore, reperfusion therapy as a percutaneous coronary intervention (PCI) or fibrinolytic therapy should be attempted as soon as possible (14). The impact of AMI localization on VPBs is also unclear. In general, large studies analyzed the occurrence of VPBs in the post infarction period by anterior or inferior localization but, the results are contradictory. Bluazite had more VPBs in inferior (15), while Stone concluded that VPBs are more frequent in anterior AMI (16) localization. Our aim was to determine the impact of AMI localization and type of reperfusion therapy on the frequency of VPBs in patients with reduced LVEF. PATIENTS AND METHODS Patients This retrospective research was conducted from the 2000 to Among the total of 705 patients with acute ST elevation myocardial infarction (STEMI) (the average age 62.5 years), 484 (68.7%) were males and 221 (31.3%) were females. They were hospitalized in the Coronary Unit of the General Hospital in Karlovac, Croatia. Methods All patients were divided according to the reperfusion therapy to patients with fibrinolysis and the PCI. According to the AMI localization, the patients were divided into four groups (anteroseptal, anterolateral, inferior and posterior localization). Because LVEF<50% represents a reduced value of LVEF, we established the experimental group of patients with LVEF<45% and the control group with better LVEF (>45%). According to the number of VPBs, we established the group with less VPBs (<10 per hour) and the group with more VPBs (>10 per hour). The exclusion criteria were cardiomyopathy, left ventricular hypertrophy, hyperkalemia, hyperthyroidism, mitral valve prolapse, digitalis therapy, history of VPBs, and previous myocardial infarctions. The AMI diagnostic criteria were chest pain (the first six hours). The ECG diagnosis of the ST segment elevation type of acute myocardial infarction require at least 1 mm (0.1 mv). These elevations must be present in anatomically contiguous leads (I, avl, V3-V6 correspond to the anterolateral wall; V1-V4 correspond to the anteroseptal wall; II, III, avf correspond to the inferior wall and ST-denivelation in V3-4 or R-wave enlargement in V1-V3 leads for posterior AMI). The cardiac enzyme troponin I was >1.00 ug/l. Streptokinase was used as a fibrinolytic agent, and the dose was 1.5 million international units. Each patient was treated with beta-blockers, acetylsalicylic acid (ASA), angiotensin converting enzyme inhibitor, statin and in the PCI method with clopidogrel. The PCI treatment had to be with stenting. 140

39 Horvat et al. VPBs in myocardial infarction A coronary angiogram in the PCI treatment had the criteria of post angioplasty blood flow of the TIMI grade III (17). Ventricular premature beats had been verified between the sixth and tenth day of hospitalization with a 24 hour Holter ECG monitoring. Left ventricular ejection fraction was assessed with the heart ultrasound and the Simpson biplane technique in the apical projection. RESULTS The study evaluated a total of 705 patients. The patients average age was 62.4 years, 484 (68.7%) were males and 221 (31.3%) were females. A significant number of VPBs were identified in 194 (27.4%) patients. A total of 155 (22.0%) patients had the LVEF<45%. A total of 166 (23.5%) patients had anteroseptal, 106 (15.0%) anterolateral, 159 (22.6%) inferior and 274 (38.9%) posterior infarct localization (Table 1). Table 1. Distribution of myocardial infarction localization Localization N (%) of patients Anteroseptal 166 (23.5) Anterolateral 106 (15.0) Inferior 159 (22.6) Posterior 274 (38.9) Total 705 (100) There were 522 patients with fibrinolysis and 183 patients with PCI treatments (Table 2). Table 2. Distribution of reperfusion therapy Reperfusion therapy N (%) of patients Fibrinolysis 522 (74) Percutaneous coronary intervention 183 (26) Total 705 (100) In the fibrinolysis and LVEF<45% group, a significant number of VPBs were in anteroseptal (p=0.017), anterolateral (p<0.001) and posterior localization (p<0.001) (Table 3). Table 3. Myocardial infarction and ventricular premature beats in fibrinolysis Localization of myocardial infarction Anteroseptal Anterolateral Inferior Posterior Ventricular premature beat/h No (%) of patients with left ventricular ejection fraction <45% >45% 95% CI p <10 28 (5.4) 72 (13.8) >10 18 (3.4) 18 (3.4) <10 14 (2.7) 52 (10.0) >10 16 (3.1) 8 (1.5) <0.001 <10 12 (2.3) 88 (16.9) >10 8 (1.5) 22 (4.2) <10 10 (1.9) 120 (23.0) >10 14 (2.7) 22 (4.2) <0.001 In the PCI treatment significant number of VPBs were in anteroseptal localization (p=0.001) (Table 4). Table 4. Myocardial infarction and ventricular premature beats in percutaneous coronary intervention Localization of myocardial infarction Anteroseptal Anterolateral Inferior Posterior DISCUSSION Ventricular premature beat/h No (%) of patients with left ventricular ejection fraction <45% >45% 95% CI p <10 2 (1.1) 20 (10.9) >10 6 (3.3) 2 (1.1) <10 2 (1.1) 8 (4.4) >10 4 (2.2) 2 (1.1) <10 4 (2.2) 14 (7.7) >10 2 (1.1) 9 (4.9) <10 6 (3.3) 60 (32.8) >10 9 (4.9) 33 (18.0) This study established several VPBs in patients with fibrinolysis than PCI treatment in acute STEMI. In relevant literature sources primary PCI was better than thrombolytic therapy (18). Primary PCI is more effective than thrombolytic therapy for the treatment of acute STEMI because it reduces overall short-term death (7% vs 9%), death excluding the shock (5% vs 7%), non-fatal reinfarction (3% vs 7%), stroke (1% vs 2%) and the combined endpoint of death, non-fatal reinfarction, and stroke (8% vs 14%) (19). However, there are scarce data about VPBs in patients with fibrinolysis comparing to PCI treatment in acute STEMI. Our results can be compared with a limited number of relevant literature sources. A higher VPBs frequency in anterior than inferior infarctions (70.2 vs 58.9%) has been described by Stone (16). Breithardt described a higher frequency of tardive ventricular potentials in anterior AMI compared to the inferior AMI (20). However, Bluzaite (15) had higher occurrence in the inferior than anterior infarction. Pascale also found a significantly more VPBs in the inferior infarction (21). Future researches with an additional electrophysiological testing would be of interest. Although the cardiac conductive system is mainly in the septum (22), the distal area like His-Purkinje system is also a source of ectopic impulses (23). An early spontaneous reperfusion in AMI could be a protective factor for VPBs (24,25). The impact of collateral circulation is also important. In case of collateral circulation, the infarction affec- 141

40 Medicinski Glasnik, Volume 12, Number 2, August 2015 ted area will be smaller than the area with occluded artery (26). Our results had the link among the residual LVEF<45% and the significant number of VPBs. These data are in correlation with results of low LVEF and several VPBs (27). Solomon demonstrated the rise in the mortality rate within 30 post-mi days in LVEF<30% (13). Our analysis verified good results in the PCI treatment. The Croatian PCI network is very important for this process. This is important because the previous large trials such as DANAMI-2 study did not verify less VPBs in the PCI than the fibrinolysis (28). In conclusion, the AMI cum small LVEF and PCI therapy has less VPBs than the fibrinolysis therapy. In the fibrinolysis therapy more VPBs were in anteroseptal, anterolateral and posterior localization but in the PCI treatment only in anteroseptal localization. In patients with reduced ejection fraction in AMI, treatment with PCI method has a better antiarrhythmic effect compared to fibrinolysis treatment. For this reason, the PCI treatment reduces the possibility of the occurrence of malignant ventricular arrhythmias and sudden death of patients. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. REFERENCES 1. Olgin JE, Zipes DP. Specific Arrhythmias: Diagnosis and Treatment. In: Braunwald, Zipes, Libby, eds. Heart Disease. 7 th ed. Philadelphia: Elsevier/Saunders, 2005: Drőgeműller A, Seidl K, Schiele R, Schneider S, Gitt A, Gotwik M. Prognostic value of non-sustained ventricular tachycardias after acute myocardial infarction in the thrombolytic era: importance of combination with frequent ventricular premature beats. Z Kardiol 2003; 92: Timmer JR, Breet N, Svilaas T, Haaksma J, Van Gelder IC, Zijlstra F. Predictors of ventricular tachyarrhythmia in high-risk myocardial infarction patients treated with primary coronary intervention. Neth Heart J 2010; 18: Volpi A, Cavalli A, Turato R, Barlera S, Santoro E, Negri E. Incidence and short-term prognosis of late sustained ventricular tachycardia after myocardial infarction: results of the Gruppo Italiano per lo Studio della Sopravvivenza nell Infarto Miocardico (GI- SSI-3) Data Base. Am Heart J 2001; 142: Newbi KH, Thompson T, Stebbins A, Topol EJ, Califf RM, Natale A. Sustained ventricular arrhythmias in patients receiving thrombolytic therapy: incidence and outcomes. Circulation 1998; 98: Breithardt G, Borggrefe M, Martinez-Rubio A, Budde T. Pathophysiological mechanisms of ventricular tachyarrhythmias. Eur Heart J 1989; 10: Manolis AS. Ventricular premature beats. uptodate.com/home (16 January 2014). 8. Ventricular premature beats. The Merck Manuals: The Merck Manual for Health Care Professionals. disorders/ventricular_premature_beats_vpb.html (16 January 2014). 9. Cha YM, Lee GK, Klarich KW, Grogan M. Premature ventricular contraction-induced cardiomyopathy. Circulation: Arrhythmia and Electrophysiology 2012; 5: Arrhythmia. National Heart, Lung, and Blood Institute. topics/arr/ (16 January 2014). 11. Eckardt L, Breithardt, G. Drug-induced ventricular tachycardia. In: Zipes DP, Jalife J, ed. Cardiac electrophysiology, From Cell to Bedside. 6 th ed. Elsevier Saunders, Philadelphia; 2014: John RM, Tedrow UB, Koplan BA, Albert CM, Epstein LM, Sweeney MO, Miller AL, Michaud GF, Stevenson WG. Ventricular arrhythmias and sudden cardiac death. Lancet 2012; 380: Solomon SD, Zelenkofske S, McMurray JJ, Finn PV, Velazqueze E, Ertl G. Sudden death in patients with myocardial infarction and left ventricular dysfunction, heart failure, or both. N Engl J Med 2005; 352: Terkelsen CJ, Christiansen EH, Sorensen JT, Kristensen SD, Lassen JF, Thuessen L. Primary PCI as the preferred reperfusion therapy in STEMI: it is a matter of time. Heart 2009; 95: Bluazite I, Brazdzionyte J, Bluzhas J, Mickeviciene A. Signal-averaged electrocardiogram peculiarities of the first and recurrent myocardial infarction. JHong Kong Coll Cardiol 1997; 5: Stone PH, Raabe DS, Jaffe AS, Gustafson N, Müller JE, Turi ZG. Prognostic significance of location and type of myocardial infarction: independent adverse outcome associated with anterior location. J Am Coll Cardiol 1998; 11: Gibson CM, Ryan KA, Kelley M, Rizzo MJ, Mesley R, Murphy S. Methodologic drift in the assessment of TIMI grade 3 flow and its implications with respect to the reporting of angiographic trial results. Am Heart J 1999; 137: Weaver WD, Simes RJ, Betriu A, Grines CL, Zijlstra F, Garcia E, Grinfeld L, Gibbons RJ, Ribeiro EE, DeWood MA, Ribichini F. Comparison of primary coronary angioplasty and intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review. JAMA 1997; 278:

41 Horvat et al. VPBs in myocardial infarction 19. Keeley EC, Boura JA, Grines CL. Primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomised trials. Lancet 2003; 361: Breithard LG, Schwarzmaier J, Borggrefe M, Haerten K, Seipel L. Prognostic significance of late ventricular potentials after acute myocardial infarction. Eur Heart J 1983; 4: Pascale P, Schlaepfer J, Oddo M, Schaller MD, Pierre Vogt P, Fromer M. Ventricular arrhythmia in coronary artery disease: limits of a risk stratification strategy based on the ejection fraction alone and impact of infarct localization. Europace 2009; 11: Christoffels VM, Moorman AFM. Why Are Some Regions of the Heart More Arrhythmogenic Than Others? Circulation: Arrhythmia and Electrophysiology 2009; 2: Dave J, Lakhia R, Jha SH.Ventricular Premature Complexes. article/ overview (15 September 2014) 24. Bainey KR, Fu Y, Wagner GS, Goodman SG, Ross A, Granger CB, Van De Werf F, Armstrong PW. Spontaneous reperfusion in ST-elevation myocardial infarction: comparison of angiographic and electrocardiographic assessments. Am Heart J 2008; 156: Bainey KR, Fu Y, Granger CB, Hamm CW, Holmes DR, O Neil WW. Benefit of angiographic spontaneous reperfusion in STEMI: does it extend to diabetic patients? Heart 2009; 95: Fujita M, Nakae I, Kihara Y, Hasegawa K, Nohara R, Ueda K. Determinants of collateral development in patients with acute myocardial infarction. Clin Cardiol 1999; 22: Schuster EH, Bulkley BH. Ischemia at a distance after acute myocardial infarction: a cause of early postinfarction angina. Circulation 1980; 62: Hofsten DE, Wachtell K, Lund B, Molgaard H, Egstrup K. Prevalence and prognostic implications of non-sustained ventricular tachycardia in ST-segment elevation myocardial infarction after revascularization with either fibrinolysis or primary angioplasty. Eur Heart J 2007; 28: Utjecaj reperfuzijske terapije i lokalizacije infarkta na učestalost ventrikulskih ekstrasistola u akutnom infarktu miokarda Davor Horvat 1, Josip Vincelj 2,3,4 1 Služba za internu medicinu, Opća bolnica Karlovac, Karlovac, 2 Zavod za bolesti srca i krvnih žila, Klinička bolnica Dubrava, Zagreb 3 Zdravstveno veleučilište, Zagreb, 4 Medicinski fakultet Osijek, Sveučilište J. J. Strossmayera, Osijek; Hrvatska SAŽETAK Cilj Odrediti utjecaj lokalizacije infarkta i reperfuzijske terapije na učestalost ventrikulskih ekstrasistola (VES) kod pacijenata s akutnim infarktom miokarda (AIM) i reduciranom ejekcijskom frakcijom lijevog ventrikla (EFLV). Metode Ukupno je 705 bolesnika s akutnim infarktom miokarda i ST elevacijom (STEMI) podijeljeno prema lokalizaciji infarkta (anteroseptalni, anterolateralni, inferiorni i posteriorni) i reperfuzijskoj terapiji (fibrinoliza ili perkutana koronarna intervencija sa stentom) u dvije grupe: EFLV<45% kao ispitivana grupa, te EFLV>45% kao kontrolna grupa. Pojava VES<10/h bila je nesignifikantna, a pojava VES>10/h signifikantna. Rezultati U pacijenata s fibrinolizom i EFLV<45% značajan broj VES-a bio je u anteroseptalnom (p=0,017), anterolateralnom (p<0,001) i posteriornom AIM-u (p<0,001), a u pacijenata s perkutanom koronarnom intervencijom (PCI) i EFLV<45% značajan broj VES-a bio je samo u anteroseptalnoj AIM (p=0,001) lokalizaciji. Zaključak U pacijenata sa smanjenom ejekcijskom frakcijom u AIM-u, tretman s PCI metodom imao je bolji antiaritmički efekat u odnosu na fibrinolitički tretman. Ključne riječi: fibrinoliza, perkutana koronarna intervencija, ventrikulska aritmija 143

42 ORIGINAL ARTICLE Possibilities of differentiation of solitary focal liver lesions by computed tomography perfusion Irmina Sefić Pašić 1, Anes Pašić 2, Spomenka Kristić 1, Adnan Beganović1, Aladin Čarovac 1, Amra Dzananovic 1, Lidija Lincender 3, Sandra Vegar Zubović 1 1 Radiology Clinic, 2 Oncology Clinic; Clinical Center of Sarajevo University, 3 Academy of Sciences and Arts of Bosnia and Herzegovina; Sarajevo, Bosnia and Herzegovina ABSTRACT Aim To evaluate possibilities of computed tomography (CT) perfusion in differentiation of solitary focal liver lesions based on their characteristic vascularization through perfusion parameters analysis. Corresponding author: Irmina Sefic Pašić Radiology Clinic, Clinical Center of University of Sarajevo Bolnička 25, Sarajevo, Bosnia and Herzegovina Phone : ; Fax : ; irmina.sefic@gmail.com Original submission: 22 May 2015; Revised submission: 06 July 2015; Accepted: 08 July doi: / Methods Prospective study was conducted on 50 patients in the period Patients were divided in two groups: benign and malignant lesions. The following CT perfusion parameters were analyzed: blood flow (BF), blood volume (BV), mean transit time (MTT), capillary permeability surface area product (PS), hepatic arterial fraction (HAF), and impulse residual function (IRF). During the study another perfusion parameter was analyzed: hepatic perfusion index (HPI). All patients were examined on Multidetector 64-slice CT machine (GE) with application of perfusion protocol for liver with i.v. administration of contrast agent. Results In both groups an increase of vascularization and arterial blood flow was noticed, but there was no significant statistical difference between any of 6 analyzed parameters. Hepatic perfusion index values were increased in all lesions in comparison with normal liver parenchyma. Conclusion Computed tomography perfusion in our study did not allow differentiation of benign and malignant liver lesions based on analysis of functional perfusion parameters. Hepatic perfusion index should be investigated in further studies as a parameter for detection of possible presence of micro-metastases in visually homogeneous liver in cases with no lesions found during standard CT protocol Key words: CT protocol, contrast media, hepatic perfusion index Med Glas (Zenica) 2015; 12(2):

43 Sefić Pašić et al. Computed tomography perfusion in liver INTRODUCTION The liver is the most common site of metastases from gastrointestinal tumors (1,2). High blood flow (about 25% of cardiac output), favorable microscopic anatomy (liver sinusoids and gaps in subendothelial basement membrane), and rich biochemical environment favor the rapid growth of metastatic deposits in the liver (1,2). In malignant diseases diagnosis of the extent of the primary tumor and staging of a potential spread of the disease have fundamental importance. Without this information, an appropriate therapy is not possible (1,2). A significant problem for all diagnostic imaging methods in the staging of malignant disease is a relatively high incidence of benign lesions (1,3). For this reason high diagnostic specificity is a major requirement in order to distinguish various benign lesions that may affect the therapeutic decisions in case of misinterpretation (1,3). In general, detection of metastases with diagnostic methods is based on micro and macrostructure changes that distinguish tumor tissue from normal liver tissue (1,2). Because many pathological conditions of the liver leading to changes in regional or whole blood flow, perfusion imaging of the liver proved to be a method with high sensitivity and specificity in differentiating of liver lesions (3,4). Kinkel at al. analyzed in meta-analysis a sensitivity of different diagnostic methods in diagnosing metastasis and showed that in liver tumors over 1 cm (sensitivity 55-90%), or for lesions less than 1 cm sensitivity is much lower (below 50%), and microscopic lesions remain occult (3). Smith et al. pointed that CT perfusion is one of the last achievements in the field of physiological imaging, which can provide new opportunities for the use of imaging as a biomarker (5). Since it was first described by Miles et al. (6), CT perfusion has been successfully applied in a variety of clinical conditions including assessment of liver cirrhosis (7), characterization of liver tumors (8,9), and evaluation of therapy response in liver diseases (9-11) The aim of this study was to analyze CT perfusion parameters (blood flow, blood volume, hepatic arterial fraction, mean transit time, capillary permeability surface area product, impulse residual function) and to determine whether one or more of the six parameters significantly stand out in differentiating pathological lesions to benign and malignant. The purpose of the study was to evaluate the possibility of the application of CT perfusion imaging in the differentiation of focal hepatic lesions based on the perfusion analysis. PATIENTS AND METHODS The prospective study included 50 patients in the period at the Radiology Clinic of Clinical Center of Sarajevo University. All patients were examined on Multidetector 64-slice CT machine (Light Speed VCT) (GE Medical Systems, Milwaukee, WI, USA) with application of CT perfusion protocol for liver with i.v. administration of contrast agent. Solitary liver characteristics changes and their division into benign and malignant were confirmed by at least two radiological methods (ultrasound, computerized tomography or magnetic resonance) with follow up of focal lesion over a certain period of time, and based on clinical parameters. All patients had previously performed an ultrasound examination of the abdomen with a special focus on the liver to verify solitary focal lesion in liver. Based on the differences in perfusion parameter results, further categorization of tumors or secondary deposits (based on histological diagnosis) into subgroups of solitary liver lesions was made. Tube voltage 120 kv, power tubes 60mA, exposure time 50 sec, thickness 0.5 cm, and the beam width 4 cm were used as an examination protocol. The amount of contrast agent that is administered was 0.5 ml/kg body weight of the patient at a flow rate 4 ml/sec. Before administration of contrast material all patients signed a consent for the application, and were informed about adverse reactions. The study was approved by the Ethics Committee of the Clinical Center of University of Sarajevo and all patients signed informed consents for inclusion in study. Six perfusion parameters were analyzed (12). Blood flow (BF) (ml/min/100 g tissue), which was carried out both in arterial and in the portal phase, is the volume of blood flow through blood vessels including large collecting blood vessels, arteries, arterioles, venules, veins and sinuses. 145

44 Medicinski Glasnik, Volume 12, Number 2, August 2015 Mean transit time (MTT) is measured in seconds. Blood moves through the blood vessels at different speed so that there is no universally defined time flow of blood from the arterial network in the vein. Distribution of flow time and MMT represent median time of that distribution. Capillary permeability surface area product (PS) (ml/min/100 g tissue) is the flow of the contrast medium through the capillary endothelium in interstitial space. Hepatic arterial fraction (HAF) (%) is the percentage of blood that supplies hepatic arteries in relation to the portal vein in the liver. Impulse residual function (IRF) (ml/min/100 g) is the ratio of arterial and interstitial concentration of the contrast medium. Functional perfusion parameters were analyzed by Deconvolution (13,14) method and compartmental model (15,16). The balance between the arterial and portal inputs was expressed by the hepatic perfusion index (HPI), which represents the ratio of arterial blood flow (Fa) and total hepatic flow Ft (Fa + Fp). Statistical analysis was done by using chi-square test, Student s t-th test, analysis of variance (ANOVA), and to determine the degree of mutual dependence (correlation) of certain parameters Spearman s rank correlation coefficient was used. Testing the sensitivity of some parameters was carried out by analyzing the area under the ROC curve (receiver operator curve). For statistical analysis of hepatic perfusion index, control group was introduced referring to the values of normal liver parenchyma, and it was compared with the values obtained in pathologic lesions (it only applies on HPI parameter) RESULTS A total of 30 women and 20 men was included in the study. Analysis by gender revealed that women were more frequently represented in the group of benign lesions, 18 (69.2%), than men, eight (30.8%), while in the group with other lesions the same number of men and women was recorded, 25 in each (50%) (p>0.05). The patients with benign lesions were (on average) slightly older (60.7 ± 9.6 years; range yr.) than the patients with other lesions (57.8 ± 13.5 years; range years) (p> 0.05). Of the 24 malignancies 10 had histological diagnosis, be it a primary tumor or a secondary deposit. Analysis of blood flow showed that patients with malignant lesions had a little higher value, but without significant statistical difference (p> 0.05), with emphasis that the significant difference was not shown among the subgroups, e.g., metastases vs. metastases with patohistological diagnosis (phd). There was no statistically significant difference found in blood volume or arterial or in the portal series (p>0.05). Values for the MTT, HAF, SF and IRF (including both arterial and portal phase), showed no statistically significant differences. Analysis of the parameters of aortic blood flow showed that the highest sensitivity in differentiating between benign and malignant lesions has shown surface permeability (53.8%), and the lowest one hepatic arterial fraction (42.9%) with no statistical differences (Figure 1). Figure 1. Specificity and sensitivity of aortic blood flow in both groups of patients (benign and malignant lesions) Analysis of the parameters of the portal system showed that the highest sensitivity in differentiating between benign and malignant lesions has shown permeability surface (56.8%), and the lowest one volume hepatic blood volume (42.3%) without statistically significant difference between individually observed parameters (Figure 2). A statistically significant correlation between the groups, e.g., benign and malignant lesions, and six perfusion parameters tested in both arterial and portal phase was not observed in any case. The highest correlation between perfusion parameters in differentiating benign from malignant lesions showed blood flow in the aortic 146

45 Sefić Pašić et al. Computed tomography perfusion in liver Figure 2. Sensitivity and specificity of portal blood flow in both groups of patients (benign and malignant lesions) blood stream (Ro = 0.137) followed by hepatic arterial fraction aortic (Ro = 0.133) and blood volume portal (Ro = 0.133), then the permeability surface - portal (Ro = ), and hepatic arterial fraction portal (Ro = 0.108) followed by other parameters with less than 10% effect on the differentiation of groups (Table 1). The patients with benign lesions had an average HPI slightly higher (55.7 ± 5.1; range 50 to 65.5%) than the patients with other lesions (54.6 ± 3.95; range 50 to 66.9 %) with no statistically significant differences (p> 0.05). Table 1. Correlations between groups (benign and malignant lesions) and between each of six perfusion parameters Correlation coefficient Benign Malignant Blood flow (ml/min/100g) - aortal Blood volume (ml/100g) - aortal Mean transit time (sec) - aortal Hepatic arterial fraction - aortal Permeability surface (ml/min/100g) - aortal Impulse residual function - aortal Blood flow (ml/min/100g) - portal Blood volume (ml/100g) - portal Mean transit time (sec) - portal Hepatic arterial fraction - portal Permeability surface (ml/min/100g) - portal Impulse residual function - portal The analysis of HPI showed that sensitivity in differentiating benign lesions from other lesions on the basis of this parameter was 8.4% (Figure 3). A comparison of HPI values in focal liver lesions (benign and malignant) with values of normal liver parenchyma has shown statistically significant difference (p <0.05). The Spearman s correlation coefficient indicated that with 82.6% certainty the patients with normal values of hepatic perfusion index in liver parenchyma belonged to the control group and Figure 3. Sensitivity and specificity of hepatic perfusion index in hypervascular benign lesions patients with elevated values belonged to the group with benign or malignant lesions. DISCUSSION Quantitative measurement of perfusion CT provides information about the processes that affect the structure and function of the tissue. The concept is based on monitoring the first pass bolus of iodinated contrast agents through blood vessels of a certain tissue. This method allows non-invasive monitoring of changes in malignant process, as well as the results of treatment, and considering that CT perfusion provides data on angiogenesis activity may be useful in monitoring the treatment of angiogenesis inhibitors (17). In patients with known metastatic disease, an elevated arterial perfusion was noticed with values of about ml/min/100 ml m versus ml/min/100 ml in the healthy control group (values of normal liver parenchyma). Therefore, HPI was significantly higher in patients with metastatic disease, which was proven by Miles et al. (18-20) and Blomley et al. (21). In our study, the value of HPI in solitary lesions ranged between 50 and 60%, with no significant differences by type of lesion. Also, benign and malignant lesions had the same value of HPI, considering that the benign lesions in this study represent only hemangiomas, which are basically hypervascular lesions. The results of this study indicated that CT perfusion is not the method of choice in the diagnosis of hemangioma, be- 147

46 Medicinski Glasnik, Volume 12, Number 2, August 2015 cause they can be diagnosed with the standard protocol that is used during the CT examination. Certainly, HPI parameter can be used to prove the presence of micrometastases in visually homogeneous liver, where a standard way of CT protocols showing no enhancement after contrast administration (4,18). Any increase in the value of HPI favors of the changes with intense vascularization, which comes from the hepatic artery, but without the possibility of characterization of these lesions, so that this parameter remains highly sensitive, but not specific enough (4,18). In rats, Cuenod et al. used deconvolution technique and found colon cancer metastasis in the liver with increased HPI and reduction in hepatic perfusion due to the reduction of portal perfusion. They also observed decrease in distribution volume and increase in MTT (18). Further sub-analyses of the two groups (benign and malignant lesions) in the present study revealed that patients with histologically verified malignant lesions had no significant differences in perfusion parameters values compared to patients with malignant change without histopathologic verification. Modification of hepatic perfusion can be found not only in patients with visible liver metastases, but also in patients with occult metastases that develop liver metastases at follow-up examinations (21). Leggett et al. described changes in hepatic perfusion in patients with visible metastases, reduction of portal perfusion and increased HPI in patients with occult metastases in whom the disease is detected at follow-up (4). Routine CT and MRI are insensitive to discover occult and early stage hepatic micrometastasis of tumors (22). Hemangioma is one of the common benign liver tumors; however, it is sometimes misdiagnosed as a malignant tumor (23). Although there is no apparent abnormality in morphology, computed tomography perfusion can display changes in hemodynamics through its functional imaging (24). An increase in both HAP and HPI can declare the possibility of liver micrometastasis (15). Cuenod et al. (13-17) used the deconvolution method to study liver hemodynamic changes caused by occult hepatic micrometastasis in rats and found micrometastases in normal liver leading to 34% decrease in portal blood flow and 25% increase in MTT, suggesting that resistance is increased in sinusoidal capillaries. Limitation of CT perfusion arises from the large number of parameters that require dual model of hepatic microcirculation. The liver perfusion models, measurements are taken during the first passing of the contrast (25). In cases of the existence of liver nodules or in chronic liver disease, where there is a modification of a sinusoidal permeability and interstitial volume, it is required to have more complex models (26,27). Little attention is devoted to biomarkers resulting from radiological examinations. CT perfusion is one of the recent developments in the field of physiological imaging, which can provide new opportunities for the use of imaging as a biomarker (28). Preliminary evidence suggests that measurement of liver perfusion can be connected with the survival of the patients with visible metastasis and patients with micrometastasis, where conventional CT protocol did not detect changes in liver parenchyma. Many cancer patients undergo CT examinations of the liver, and consequently, recurrent tumors are identified after a primary treatment. For colorectal carcinomas, intensified follow-up in this way is associated with decreased mortality (29) and the American Association of Clinical Oncologists now recommends annual CT examinations of the lungs and abdomen in the first three years after the primary therapy in patients with high risk of recurrent disease (30). CT perfusion could be incorporated into such programs. CT perfusion is especially suitable for the assessment of the response to biological therapy, which affects the tumor blood vessels, giving quantitative information, and more importantly, studies have shown that obtained perfusion parameters correlate with histological measurements of angiogenesis (31-34). The possibility of identifying high risk of liver metastases in common diseases such as colorectal cancer can help with decisions on adjuvant chemotherapy, but also can avoid unnecessary treatment of patients who are at low risk of developing liver metastases (9,34). Computed tomography is the most common modality for evaluating cancer patients. In our study CT perfusion did not allow differentiation between benign and malignant focal liver lesions. However, it is possible that larger patient population should be studied. CT perfusion can be ea- 148

47 Sefić Pašić et al. Computed tomography perfusion in liver sily included as a part of standard CT protocol in order to provide functional information about the solitary change. It is an available method, easy to perform, allows repeated examinations and applicable to all organic systems. REFERENCES 1. Leen E. The detection of occult liver metastases of colorectal carcinoma. J Hepatobiliary Pancreat Surg 1999; 6: Seto S, Onodera H, Kaido T, Yoshikawa A,Ishigami S, Arii S, Imamura M. Tissue factor expression in human colorectal carcinoma: correlation with hepatic metastasis and impact on prognosis. Cancer 2000; 88: Kinkel K, Lu Y, Both M, Warren RS, Thoeni RF. Detection of hepatic metastases from cancers of the gastrointestinal tract by using noninvasive imaging methods (US, CT, MR imaging, PET): a meta-analysis. Radiology 2002; 224: Miles KA, Hayball MP, Dixon AK. Functional images of hepatic perfusion obtained with dynamic CT. Radiology 1993; 188: Smith JJ, Sorensen AG, Thrall JH. Biomarkers in imaging: realizing radiology s future. Radiology 2003; 227: Miles KA, Hayball M, Dixon AK. Colour perfusion imaging : a new application of computed tomography. Lancet 1993; 337: Ronot M, Asselah T, Paradis V, Michoux N, Dorvillius M, Baron G, Marcellin P, Van Beers BE, Vilgrain V. Liver fibrosis in chronic hepatitis C virus infections: differentiating minimal from intermediate fibrosis with perfusion CT. Radiology 2010; 256: Sahani DV, Holalhere NS, Mueller PR, Zhu AX. Advanced hepatocellular carcinoma: CT perfusion of liver and tumor tissue-initial experience. Radiology 2007; 243: Hayano K, Desai GS, Kambadakone R, Fuentes JM, Tanabe KT, Sahani DV. Quantitative characterisation of hepatocellular carcinoma and metastatic liver tumor by CT perfusion. Cancer Imaging 2013; 13: Ippolito D, Capraro C, Casiraghi A, Cestari C, Sironi S. Quantitative assesment of tumour associated neovascularisation in patients with liver cirrhosis and hepatocellular carcinoma : role of dynamic-ct perfusion imaging. Eur Radiol 2012; 22: Jiang T, Kambadakone A, Kuikarni NM, Zhu AX, Sahani DV. Monitoring response to antiangiogenic treatment and predicting outcomes in advanced hepatocellular carcinoma using image biomarkers, CT perfusion, tumor density and tumor size (RECIST). Invest Radiol 2012; 47: Miles KA. Measurement of tissue perfusion by dynamic computed tomography. Br J Radiol 1991; 64: Cuenod C, Leconte I, Siauve N, Resten A, Dromain C, Poulet B, Frouin F, Clément O, Frija G. Early changes in liver perfusion caused by occult metastases in rats: detection with quantitative CT. Radiology 2001; 218: FUNDING No specific funding was received for this study TRANSPARENCY DECLARATION Competing interests : None to declare 14. Fournier LS, Cuenod CA, de Bazelaire C, Siauve N, Rosty C, Tran PL. Early modifications of hepatic perfusion measured by functional CT in a rat model of hepatocellular carcinoma using a blood pool contrast agent. Eur Radiol 2004; 14: Materne R, Van Beers BE, Smith AM, Leconte I, Jamart J, Dehoux JP, Keyeux A, Horsmans Y. Non-invasive quantification of liver perfusion with dynamic computed tomography and a dual-input onecompartmental model. Clin Sci (Lond) 2000; 99: Van Beers BE, Leconte I, Materne R,Smith AM, Jamart J, Horsmans Y. Hepatic perfusion parameters in chronic liver disease: dynamic CT measurements correlated with disease severity. AJR Am J Roentgenol 2001; 176: Pandharipande Pari V, Krinsky Glenn A., Rusinek H., Lee Vivian S. Perfusion imaging of the liver: current challenges and future goals. Radiology 2005; 234: Dugdale PE, Miles KA. Hepatic metastases: the value of quantitative assessment of contrast enhancement on computed tomography. Eur J Radiol 1999; 30: Miles KA, Leggett DA, Kelley BB, Hayball MP, Sinnatamby R, Bunce I. In vivo assessment of neovascularization of liver metastases using perfusion CT. Br J Radiol 1998; 71: Miles KA, Kelley BB. Altered perfusion adjacent to hepatic metastases. Clin Radiol 1997; 52: Blomley M, Coulden R, Dawson P. Liver perfusion studied with ultrafast CT. J Comput Assist Tomogr 1995; 19: Kinkel K, Lu Y, Both M, Warren RS, Thoeni RF. Detection of hepatic metastases from cancers of the gastrointestinal tract by using noninvasive imaging methods (US, CT, MR imaging, PET): a meta-analysis. Radiology 2002; 224: Semelka RC, Sofka CM.Hepatic hemangiomas. Magn Reson Imaging Clin N Am 1997; 5: Smith JJ, Sorensen AG, Thrall JH. Biomarkers in imaging: realizing radiology s future. Radiology 2003; 227: Cuenod CA, Leconte I, Siauve N, Frouin F, Dromain C, Clément O, Frija G. Deconvolution technique for measuring tissue perfusion by dynamic CT: application to normal and metastatic liver. Acad Radiol 2002; 9: S Kapanen MK, Halavaara JT, Hakkinen AM. 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48 Medicinski Glasnik, Volume 12, Number 2, August Smith JJ, Sorensen AG, Thrall JH. Biomarkers in imaging: realizing radiology s future. Radiology 2003; 227: Jeffrey GM, Hickey BE, Hider P. Follow-up strategies for patients treated for non-metastatic colorectal cancer (Cochrane Review). In: The Cochrane Library. Issue 2. Oxford: Update Software, Desch CE, Benson AB 3rd, Somerfield MR, Flynn PJ, Krause C, Loprinzi CL, Minsky BD, Pfister DG, Virgo KS, Petrelli NJ. Colorectal cancer surveillance: 2005 update of an American Society of Clinical Oncology practice guideline. J Clin Oncol 2005; 23: Hayashi K, Tozaki M, Sugisaki M, Yoshida N, Fukuda K,Tanabe H. Dynamic multislice helical CT of ameloblastoma and odontogenic keratocyst: correlation between contrast enhancement and angiogenesis. J Comput Assist Tomogr 2002; 26: Jinzaki M, Tanimoto A, Mukai M, Ikeda E, Kobayashi S,Yuasa Y, Narimatsu Y, Murai M. Double-phase helical CT of small renal parenchymal neoplasms: correlation with pathologic findings and tumor angiogenesis. J Comput Assist Tomogr 2000; 24: Wang ZQ, Li JS, Lu GM et al. Correlation of CT enhancement, tumor angiogenesis and pathologic grading of pancreatic carcinoma. World J Gastroenterol 2003; 9: Zhong L, Wang WJ, Xu JR. Clinical application of hepatic CT perfusion.world J Gastroenterolol 2009; 15: Mogućnost diferencijacije solitarnih fokalnih jetrenih lezija putem perfuzije kompjuteriziranom tomografijom Irmina Sefić Pašić 1, Anes Pašić 2, Spomenka Kristić 1 Adnan, Beganović1, Aladin Čarovac 1, Amra Džananovic 1, Lidija Lincender 3, Sandra Vegar Zubović 1 1 Klinika za radiologiju; Univerzitetski klinički centar Sarajevo, 2 Klinika za onkologiju; Univerzitetski klinički centar Sarajevo, 3 Akademija nauka i umjetnosti Bosne i Hercegovine; Sarajevo, Bosna i Hercegovina SAŽETAK Cilj Utvrditi mogućnosti perfuzije kompjuteriziranom tomografijom u diferencijaciji solitarnih fokalnih lezija jetre na osnovu njihove karakteristične vaskularizacije, a putem analize parametara perfuzije. Metode Prospektivna studija je obuhvatila 50 pacijenata koji su pregledani u periodu od do godine. Pacijenti su podijeljeni u dvije grupe, odnosno u grupu s benignim i malignim lezijama jetre. Analizirano je šest parametara CT perfuzije: protok krvi (BF), volumen krvi (BV), produkt kapilarne površne permeabilnosti (PS), jetrena arterijska frakcija (HAF) i impulsna rezidualna frakcija (IRF). Tokom studije analiziran je dodatni parametar perfuzije, jetreni perfuzioni indeks (HPI). Svi pacijenti pregledani su na multidetektorskom 64-slojnom CT aparatu (GE) uz primjenu protokola za perfuziju jetre i uz i.v. aplikaciju kontrastnog sredstva. Rezultati Kod obje grupe pacijenata dokazana je povećana vaskularizacija i povišen arterijski protok, ali nije utvrđena signifikantna razlika između analiziranih šest parametara perfuzije. Vrijednosti HPI-a bile su povišene kod svih lezija u komparaciji s normalnim jetrenim parenhimom. Zaključak Perfuzija kompjuteriziranom tomografijom u našoj studiji nije omogućila diferencijaciju benignih i malignih lezija na osnovu analize funkcionalnih parametara perfuzije. Jetreni perfuzioni indeks trebao bi se u budućim studijama ispitati kao parametar za otkrivanje potencijalnog prisustva mikrometastaza u vizuelno homogenoj jetri u slučajevima kada se lezije nisu otkrile tokom pregleda standardnim CT-protokolom. Ključne riječi: CT-protokol, kontrastno sredstvo, jetreni perfuzioni indeks 150

49 ORIGINAL ARTICLE Familial autoimmune thyroid disease and PTPN-22 Gabriel Conzuelo Rodríguez 1, Hugo Mendieta Zerón 2 1 Faculty of Medicine, Autonomous University of the State of Mexico (UAEMex), 2 Asociación Científica Latina (ASCILA) and Ciprés Grupo Médico (CGM); Toluca, Estado de México, México. ABSTRACT Aim Autoimmune thyroid disease (AITD) is a multifactorial disease with a genetic predisposition. The protein tyrosine phosphatase-22 (PTPN-22) gene is a powerful inhibitor of T-cell activation. The aim of this study was to compare messenger RNA (mrna) PTPN22 expression between healthy persons and patients with hypothyroidism and with their affected relatives. Corresponding author: Hugo Mendieta Zerón Felipe Villanueva Sur 1209 Col. Rancho Dolores CP Toluca, Estado de México, México Phone/fax: ; E- mail: mezh_74@yahoo.com Original submission: 29 December 2014; Revised submission: 08 June 2015; Accepted: 02 July doi: / Methods This was a cross-sectional, prospective and descriptive study. DNA was extracted from leukocytes (4,000-10,000 cells) using the Magna Pure LC 2.0 Instrument and MagNA Pure LC RNA Isolation Kit I (Roche, Germany). A real-time polymerase reaction (qpcr) was performed utilizing the primer sets specific for the PTPN-22 gene, and the succinate dehydrogenase complex, the subunit A, Flavoprotein (Fp) (SDHA) constitutive gene. All reactions were performed with the 7500 Fast Real Time PCR System (Applied Biosystems, Applera International, Inc. Cheshire, UK) employing the SYBR Advantage qpcr Premix Kit (Clontech, USA). Results Twenty five patients with AITD (hypothyroidism), all females (mean age 39.6 ± 11.8 years) and 23 control subjects (mean age 24.4 ± 4.2 years) were included in the study. There was no statistical difference between both groups in PTPN-22 mrna expression (p = 0.125). Conclusion There is no clear difference in mrna PTPN-22 expression. The ideal genes for a systematic screening for familial AITD are yet to be found. Key words: genetic screening, hypothyroidism, qpcr Med Glas (Zenica) 2015; 12(2):

50 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION Autoimmune thyroid disease (AITD) is the most common autoimmune condition, affecting approximately 2% of the female population and 0.2% of the male population (1). Although the exact etiology is not yet known, AITD is multifactorial in that a genetic predisposition combines with environmental risk factors to promote disease (2,3). Up to 90% of patients with AITD-related hypothyroidism are anti-thyroid peroxidase (TPO) antibody-positive. It should be noted that 10 15% of the general population are positive for anti-tpo antibodies and that low titers are less specific for AITD (2). Symptoms of hypothyroidism may be subtle, even with marked biochemical derangement, but as the disease progresses, subclinical and then clinical hypothyroidism appears. In the early stages of hypothyroidism, the thyroid stimulating hormone (TSH) may be normal and anti-thyroperoxidase (TPO) antibodies may be positive with or without goiter. Later, TSH elevation becomes modest (5 10 IU/ml) with a normal FT4 (biochemical or subclinical hypothyroidism). Some studies have reported that siblings of persons affected by Graves disease or hypothyroidism have a 33% chance of developing the disease (4). In the casuistry of Ciprés Grupo Médico (CGM), Toluca, Mexico, the percentage of mothers with AITD and with an affected daughter is 13% (5). The first gene found to be associated with both Graves disease and hypothyroidism was HLA- DR3. Since this discovery, significant progress has been made in the genetic contributions and the mechanisms underlying thyroid autoimmunity. To date, several loci have been associated with AITD. In addition to HLA-DR subtypes, these include two groups of the non-major histocompatibility complex (MHC) genes, e. g., immunoregulatory genes (CD40, CTLA-4, PTPN-22, FOXP3, and CD25), and thyroid-specific genes (Tg and TSHR) (6,7). Polymorphic variations of all these genes have been identified and linked with AITD susceptibility, but the existing studies have often given inconsistent results, with some showing associations and others not (8,9). One of the many unexpected findings of these genetic studies is that the majority of the genes identified exert very minor effects (10). Indeed, with the exception of the DRb1-Arg74 HLA variant, which resulted in an Odds ratio (OR) for Graves disease of >5, all of the remaining AITD genes gave very low OR of <1.5 (11); on the other hand, family history is positive in about 50% of patients with AITD. It is usually supposed that a strong genetic effect in the disease is related with the inheritance of many genes with small effect (12). Lymphoid tyrosine phosphatase (Lyp) encoded by protein tyrosine phosphatase-22 (PTPN-22) gene locus on chromosome 1p , such as CTLA-4, is a powerful inhibitor of T-cell activation (13). A single nucleotide polymorphism in PTPN-22 has been reported as an autoimmune susceptibility locus that associates with type 1 diabetes (14), rheumatoid arthritis (15), systemic lupus erythematosus (16), Hashimoto s thyroiditis (17), Graves disease (18), Addison s disease (19), Myasthenia gravis (20), vitiligo (21), systemic sclerosis (22), and juvenile idiopathic arthritis (23), suggesting that allelic variants of PTPN-22 could predispose to a more general autoimmune diathesis. Protein tyrosine phosphatase and protein tyrosine kinases (PTKs) are enzymes that specifically catalyze the reversible addition or release of phosphate groups from tyrosine residues on signaling intermediates. Broadly speaking, PTK amplify signals, while the mode, tempo and duration of the signal are governed by PTP. Protein tyrosine phosphatase and PTK are divided into two groups: receptor (membrane bound-rptp or RPTK) or non-receptor (cytoplasmic-nrptp or NRPTK) (24). Aim of this study was to compare mrna PTPN- 22 expression between healthy persons and patients with AITD. PATIENTS AND METHODS Study population This was a cross-sectional prospective and descriptive study conducted at the Medical Sciences Research Center (CICMED), Autonomous University of the State of Mexico (UAEMex) and at the Ciprés Grupo Médico (CGM), both in Toluca, Mexico, from August 2013 to July Diagnosis of thyroidopathy was made based on the 152

51 Conzuelo Rodríguez et al. Familial thyroidopathy and PTPN-22 presence of a thyroid profile with TSH 10 IU together with a significant titer of autoantibodies (anti-thyroglobulin > 40 IU or anti-tpo > 35 IU). The study was approved by the Institutional Review Board of the Medical Sciences Research Center (CICMED), UAEMex (25/09/13) and was performed according to the ethical standards of the Helsinki Declaration (Fortaleza, Brazil). Written informed consents were obtained from all patients and their relatives who participated in this project. Sample calculation Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test, 25 subjects per group were required for the recognition as statistically significant of a difference ± 4 relative units (RU). The common standard deviation was assumed to be 5. Clinical measurements Weight (kg), height (m) (Seca, GmbH, Germany) and waist circumference (cm) were measured in all participants. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. RNA extraction A blood sample (vacutainer tubes) was taken from each patient. Leukocytes were obtained according to the ACK-lysing buffer (LONZA) protocol. Briefly, a peripheral blood sample was placed in an EDTA tube and then centrifuged at 2,500 rpm for 10 min. All samples were maintained at -80 C until further analysis. The RNA was extracted from leukocytes (4,000-10,000 cells) in the Magna Pure LC 2.0 Instrument using the MagNA Pure LC RNA Isolation Kit I (Roche, Germany). After extraction, the RNA was quantified using a Nano Photometer (Implen GmbH, Germany), reporting concentration (in μg/ ml) and purity (as 260/280 absorbance). Gene expression A total of ng total mrna was reversetranscribed into complementary RNA (cdna) utilizing a Transcriptor High Fidelity cdna Synthesis Kit (Roche Applied Science). The amount of extracted RNA was quantified by measuring the absorbance at 260 nm. The purity of the RNA was assessed according to the ratio of the absorbance values at 260 and 280 nm; purity ranged between 1.8 and 2.1, demonstrating a high RNA quality. The samples were measured with a NanoPhotometer (Implen GmbH, Germany), and the extracts were then adjusted to a concentration of 20 μg RNA L 1 for the PCR reaction. A real-time Polymerase chain reaction (qpcr) was performed using the primer sets specific for the PTPN-22 (NM_ ) as follows: forward: 5 -AGGCAGA- CAAAACCTATCCTACA-3, reverse: 5 -TGGGTGGCAATATAAGCCTTG-3 and the succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA) constitutive gene (NM_004168) as follows:, forward: 5 -AGAGGGAGGCATTCTCATTAAC-3, reverse: 5 -ACCGAGACACCACATCTCTA-3. All reactions were performed with the 7500 Fast Real Time PCR System (Applied Biosystems, Applera International, Inc., Cheshire, UK) using the SYBR Advantage qpcr Premix Kit (Clontech). The expression levels of the genes were examined by placing 4 μl of the reverse transcription mix for each PCR reaction in a total volume of 20 μl. The thermal cycling conditions were as follows: 10 min at 95 C followed by 45 cycles of denaturation at 95 C for 15 sec and annealing/extension at 60 C for 1 min. The comparative threshold cycle (CT) method was used to calculate fold amplification, as specified by the manufacturer. The Taguchi method was employed to set the best conditions for primer amplification. The fold change in PTPN-22 was normalized against the constitutively expressed reference gene and then compared with the controls (healthy volunteers) as follows: 2 ΔΔCT, where ΔΔCT=(CT-target CT-reference) treated-sample (CT-target CTreference) calibrator-sample. Calibrator-sample refers to the expression level (1 ) of the target gene normalized to the constitutive gene. The calibrator was chosen from healthy volunteers and was given a relative expression value of 1. Statistical analysis Statistical analysis was performed using the Mann Whitney U-test after performing the Levene test. The normality hypothesis was tested using the Kolmogorov- Smirnov test. Statistical significance was tested at the p 0.05 level. 153

52 Medicinski Glasnik, Volume 12, Number 2, August 2015 RESULTS Twenty five patients with AITD (hypothyroidism), all females (mean age, 39.6 ± 11.8 years) and 23 control subjects (mean age, 24.4 ± 4.2 years) were included in the study. In Table 1, we depict the affected relatives of the patients, which included one case for a grandmother, nine cases for a mother, one for a father, seven for a sister, one for a brother, four for an aunt, one for an uncle, and six for a cousin. Average time of disease evolution was 18.4 ± 24.2 months prior to participation in this study with a mean Levothyroxine dose of 66.2 ± 23.3 μg per day. Table 1. Characteristics of thirty three affected relatives in 25 patients with autoimmune hypothyroidism Case Grandmother Mother Father Sister Brother Aunt Uncle Cousin 1 X* 2 X* 3 X* 4 X 5 X 6 X*, X (3) 7 X 8 X 9 X 10 X 11 X X X* 12 X X 13 X 14 X* 15 X 16 X X (2) 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X* *maternal, paternal Anthropometrically, while the patients showed a BMI of 24.7 ± 2.4, the control group showed one of 25.1 ± 1.7. According to the place of residence there were 12 patients from Toluca, five from Mexico City, four from Metepec, and one patient from each Otzolotepec, Naucalpan, Zinacantepec, and Guadalajara. In relation to antibodies, six patients were positive for anti-thyroglobulin, five for anti-tpo, and 14 for both. In the qpcr analysis there was no statistically significant difference in PTPN-22 expression between both groups (p=0.125). In the subgroup of pati- ents with affected relatives, the 50th percentile of PTPN-22 expression was 1.13 RU (Figure 1). Figure 1. DISCUSSION The comparable prevalence and incidence of AITD in geographically different populations suggest a significant genetic effect (19,20). In this regard, the purpose of this study was not to associate a geographical location with AITD, due to lack of information related with this topic in Latin America. In another line, obesity has been postulated as a factor linked to the cause of AITD (21). Although this is a strong argument, in our sample population this possible link does not play a critical role as the patients maintained a normal BMI. In all patients with associated diseases, AITD is usually detected in its initial phase when thyroid function is preserved, with normal or only slightly elevated TSH levels. At this stage, signs and symptoms of thyroid disease are usually absent, but because worsening of thyroid function is a possibility, early recognition of thyroid dysfunction is necessary to prevent the negative effects of hypothyroidism on growth and metabolic function (16). In our study, we evaluated patients who had been already diagnosed. Subclinical thyroid disease is a common clinical problem, and because the majority of patients are asymptomatic, screening is the only way to detect the condition in most patients (22). In our study, we performed an initial familial survey without finding a clear difference in PTPN- 22 mrna expression. This is in accordance with Inoue et al. whose authors only found an association of CD40 and FCRL3 gene polymorphisms with Graves disease intractability, and a ZFAT 154

53 Conzuelo Rodríguez et al. Familial thyroidopathy and PTPN-22 polymorphism association with Hashimoto s disease severity, but neither PTPN C/G nor PTPN22 SNP37 were associated with any option (23). Contrariwise, Ichimura et al. found an association of the SNP37 of the PTPN-22 gene with susceptibility to Graves disease in a Japanese population (24). In an initial attempt, a difference in PTPN-22 expression between familiar hypothyroidism and healthy subjects cannot be concluded from our results. In contrast, several other genes have been tested without a definitive conclusion (25-26) but of these genes taken together probably do not explain more than about 10% of the heritability of AITD (27). We cannot exclude two kinds of bias in our study: recall bias and lead-time bias. In the first case, we registered four patients with affected relatives who initially denied knowing another relative suffering from hypothyroidism until a second clinical interview. In the second type of bias, it is possible that, with a different evolution time with biochemical hypothyroidism, PTPN- 22 expression could exhibit different patterns. Unfortunately, our sample size limits us to perform a stratified analysis. Expecting a lower RU difference between healthy subjects and families affected with AITD implies a higher number of persons per group to be analyzed. The question pertaining to how to explain the familial heritability may be more straightforward. First, the female preponderance is explained partially by fetal microchimerism and X-chromosome inactivation (27). Second, imprinted genes and epistasis, the modification of expression of one gene by one or several other genes, are believed to be important genetic contributors to complex diseases. We could not forget that in REFERENCES 1. Saravanan P, Dayan CM. Thyroid autoantibodies. Endocrinol Metab Clin North Am 2001; 30: Cappa M, Bizzarri C, Crea F. Autoimmune thyroid diseases in children. J Thyroid Res 2010; 2011: Hasham A, Tomer Y. Genetic and epigenetic mechanisms in thyroid autoimmunity. Immunol Res 2012; 54: Jacobson EM, Tomer Y. The CD40, CTLA-4, thyroglobulin, TSH receptor, and PTPN22 gene quintet and its contribution to thyroid autoimmunity: back to the future. J Autoimmun 2007; 28: Mendieta Zerón H. Tiroidopatía autoinmune. II Congreso Internacional de Inmunología, Estudiantes de addition to the gene gene interactions, the clinical phenotype of affected individuals is also influenced by gene environment interactions (28). In summary, the results of our study indicate that the ideal genes for systematic AITD screening continue to be missing, but these must be defined in order to be carried out in relatives of patients with AITD, including the offspring of these patients. PTPN-22 remains a possible gene candidate for further study including its promiscuous association with familial AITD, but it would be better to study a set of genes. Such a determination would have huge implications in our current screening strategies for diagnosing earlier novel thyroidopathies. Admittedly, the sample size of our family collection remained insufficiently large for conclusion of and exclusion of the participation of PTPN-22 in the genesis of AITDs in Mexican population, but we must take into account that studies in different geographic regions revealed ethnic differences in associations most probably due to founder effects and/or to the presence or absence of certain variants in specific ethnic groups (4-8). Further studies including proteomic analyses are required. ACKNOWLEDGMENTS Authors are grateful for the collaboration of José Meneses-Calderón, MD, for his support in sending three patients for genetic evaluation, and Maggie Brunner, MA, for the English style correction. FUNDING This work was partially funded by Ciprés Grupo Médico (CGM). TRANSPARENCY DECLARATIONS Conflict of interest: none to declare. Medicina Pro Investigación y I Encuentro Nacional Semilleros de la Investigación. 6 de mayo Toluca, México. 6. Ban Y, Greenberg DA, Concepción E, Skrabanek L, Villanueva R, Tomer Y. Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease. Proc Natl Acad Sci U S A. 2003; 100: Yin X, Latif R, Bahn R, Tomer Y, Davies TF. Influence of the TSH receptor gene on susceptibility to Graves disease and Graves ophthalmopathy. Thyroid 2008; 18:

54 Medicinski Glasnik, Volume 12, Number 2, August Du L, Yang J, Huang J, Ma Y, Wang H, Xiong T, Xiang Z, Zhang Y, Huang J. The associations between the polymorphisms in the CTLA-4 gene and the risk of Graves disease in the Chinese population. BMC Med Genet 2013; 14: Kahles H, Ramos-Lopez E, Lange B, Zwermann O, Reincke M, Badenhoop K. Sex-specific association of PTPN T with type 1 diabetes but not with Hashimoto s thyroiditis or Addison s disease in the German population. Eur J Endocrinol 2005;153: Ban Y, Tomer Y. Susceptibility genes in thyroid autoimmunity. Clin Dev Immunol 2005; 12: Ban Y, Davies TF, Greenberg DA, Concepcion ES, Osman R, Oashi T, Tomer Y. Arginine at position 74 of the HLA-DR beta1 chain is associated with Graves disease. Genes Immun 2004; 5: Ban Y, Greenberg DA, Davies TF, Jacobson E, Concepcion E, Tomer Y. Linkage analysis of thyroid antibody production: evidence for shared susceptibility to clinical autoimmune thyroid disease. 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Contribution of the R620W polymorphism of protein tyrosine phosphatase non-receptor 22 to systemic lupus erythematosus in Poland. Clin Exp Rheumatol 2008; 26: Criswell LA, Pfeiffer KA, Lum RF, Gonzales B, Novitzke J, Kern M, Moser KL, Begovich AB, Carlton VE, Li W, Lee AT, Ortmann W, Behrens TW, Gregersen PK. Analysis of Families in the Multiple Autoimmune Disease Genetics Consortium (MADGC) Collection: the PTPN22 620W Allele Associates with Multiple Autoimmune Phenotypes. Am J Hum Genet 2005; 76: Skorka A, Bednarczuk T, Bar-Andziak E, Nauman J, Ploski R. Lymphoid tyrosine phosphatase (PTPN22/ LYP) variant and Graves disease in a Polish population: association and gene dose-dependent correlation with age of onset. Clin Endocrinol 2005; 62: Skinningsrud B, Husebye ES, Gervin K, Løvås K, Blomhoff A, Wolff AB, Kemp EH, Egeland T, Undlien DE. Mutation screening of PTPN22: association of the 1858T-allele with Addison s disease. Eur J Hum Genet 2008; 16: Vandiedonck C, Capdevielle C, Giraud M, Krumeich S, Jais JP, Eymard B, Tranchant C, Gajdos P, Garchon HJ. Association of the PTPN22*R620W polymorphism with autoimmune myasthenia gravis. Ann Neurol 2006; 59: Dayan CM, Daniels GH. Chronic autoimmune thyroiditis. N Engl J Med 1996; 335: Díaz-Gallo LM, Gourh P, Broen J, Simeon C, Fonollosa V, Ortego-Centeno N, Agarwal S, Vonk MC, Coenen M, Riemekasten G, Hunzelmann N, Hesselstrand R, Tan FK, Reveille JD, Assassi S, García-Hernández FJ, Carreira P, Camps MT, Fernández-Nebro A, de la Peña PG, Nearney T, Hilda D, González-Gay MA, Airo P, Beretta L, Scorza R, Herrick A, Worthington J, Pros A, Gómez-Gracia I, Trapiella L, Espinosa G, Castellvi I, Witte T, de Keyser F, Vanthuyne M, Mayes MD, Radstake TR, Arnett FC, Martin J, Rueda B. Analysis of the influence of PTPN22 gene polymorphisms in systemic sclerosis. Ann Rheum Dis 2011; 70: Viken MK, Amundsen SS, Kvien TK, Boberg KM, Gilboe IM, Lilleby V, Sollid LM, Førre OT, Thorsby E, Smerdel A, Lie BA. Association analysis of the 1858C>T polymorphism in the PTPN22 gene in juvenile idiopathic arthritis and other autoimmune diseases. Genes Immun 2005; 6: Prentice LM, Phillips DI, Sarsero D, Beever K, Mc- Lachlan SM, Smith BR. Geographical distribution of subclinical autoimmune thyroid disease in Britain: a study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinol 1990; 123: Burn GL, Svensson L, Sanchez-Blanco C, Saini M, Cope AP. Why is PTPN22 a good candidate susceptibility gene for autoimmune disease? FEBS Lett 2011; 585: McGrogan A, Seaman HE, Wright JW, de Vries CS. The incidence of autoimmune thyroid disease: a systematic review of the literature. Clin Endocrinol 2008; 69: Duntas LH, Biondi B. The interconnections between obesity, thyroid function, and autoimmunity: the multifold role of leptin. Thyroid 2013; 23: Cooper DS, Biondi B. Subclinical thyroid disease. Lancet. 2012;379: Inoue N, Watanabe M, Yamada H, Takemura K, Hayashi F, Yamakawa N, Akahane M, Shimizuishi Y, Hidaka Y, Iwatani Y. Associations between autoimmune thyroid disease prognosis and functional polymorphisms of susceptibility genes, CTLA4, PTPN22, CD40, FCRL3, and ZFAT, previously revealed in genome-wide association studies. J Clin Immunol 2012; 32: Ichimura M, Kaku H, Fukutani T, Koga H, Mukai T, Miyake I, Yamada K, Koda Y, Hiromatsu Y. Associations of Protein tyrosine phosphatase nonreceptor 22 (PTPN22) gene polymorphisms with susceptibility to Graves disease in a Japanese population. Thyroid 2008; 18: Wiersinga WM. Thyroid autoimmunity. Endocr Dev 2014; 26: Balazs C. The role of hereditary and environmental factors in autoimmune thyroid diseases. Orv Hetil 2012; 153: Effraimidis G, Wiersinga WM. 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55 ORIGINAL ARTICLE Methicillin-resistant S. aureus (MRSA), extended-spectrum (ESBL)- and plasmid-mediated AmpC ß-lactamase -producing Gram-negative bacteria associated with skin and soft tissue infections in hospital and community settings Selma Uzunović 1, Branka Bedenić 2,3, Ana Budimir 3, Amir Ibrahimagić 1, Farah Kamberović 4, Zlatko Fiolić 5, Michelle I. A. Rijnders 6, Ellen E. Stobberingh 6 1 Department of Laboratory Diagnostics, Cantonal Public Health Institute of Zenica-Doboj Canton, Bosnia and Herzegovina; 2 School of Medicine, University of Zagreb, 3 Department of Molecular Microbiology, Clinical Hospital Center Zagreb; Croatia, 4 Microbiology Department, Biotechnical Faculty, University of Ljubljana, Slovenia, 5 Department of Surgery, Clinical Hospital Center Zagreb, Croatia, 6 Department of Medical Microbiology, School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Center, Mastricht, The Netherlands ABSTRACT Aim To investigate the characteristics of meticillin-resistant S. aureus (MRSA), extended-spectrum (ESBL), and plasmid-mediated AmpC beta-lactamase producing Gram-negative bacteria causing skin and soft tissue infections (SSTIs) in hospital and outpatient settings of Zenica-Doboj Canton, Bosnia and Herzegovina. Corresponding author: Selma Uzunović Department of Laboratory Diagnostics, Cantonal Public Health Institute of Zenica- Doboj Canton Fra Ivana Jukića 2, Zenica, Bosnia and Herzegovina Phone: ; Fax: ; selma_kamb@yahoo.com Original submission: 28 April 2015; Revised submission: 25 May 2015; Accepted: 19 June doi: / Methods Antibiotic susceptibility was determined by disc-diffusion and broth microdillution methods according to CLSI guidelines. MecA gene was detected by PCR, and genetic characterization of MRSA was performed using spa-typing and the algorithm based upon repeat patterns (BURP). Double-disk-synergy test was used to screen for ESBLs. PCR was used to detect bla ESBL alleles. Genetic relatedness of the strains was tested by PFGE. Results Seventeen in-patients with MRSA, 13 with ESBL-producing Gram-negative bacteria and three patients co-infected with both, were detected. Five MRSA and 16 ESBL-producing Gramnegative bacteria were found in outpatient samples. Klebsiella spp. was isolated in 11 in- and seven outpatients. MLST CC152 was the most prevalent MRSA. Seven (38.9%) Klebsiella spp. yielded amplicons with primers specific for SHV, TEM-1 and CTX- M group 1 β-lactamases. Eight K. pneumonia (44.4%) and 16 (64%) MRSA (including the in- and outpatient) strains were clonally related. Conclusion The presence of MRSA and ESBL-producing organisms causing SSTIs in the community poses a substantial concern, due to the high morbidity and mortality associated with possible consequent hospital infections. Key words: surgical wound infections, CTX-M beta-lactamase, MLST CC152, antibiotic resistance Med Glas (Zenica) 2015; 12(2):

56 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION According to Edelsberg s classification skin and soft tissue infections (SSTIs) include eighteen types of infections (1). With regard to predominanted (microbial etiology) pathogens and risk of mortality (severity of local and systemic signs) there are superficial SSTIs caused by Staphylococcus aureus or Streptococcus pyogenes, deeper or healthcare-associated infections caused by anaerobic or gram-negative organisms, and gangrenous or necrotizing infections (or often fatal infections ) (1,2). The practice guidelines of the Infectious Diseases Society of America (IDSA) for the diagnosis and management of skin and soft tissue infections (3) classifies SSTIs into five categories, including superficial, uncomplicated infection (impetigo, erysipelas and cellulitis), necrotizing infection, infections associated with bites and animal contact, surgical site infections and infections in the immunocompromised host. The purpose of all SSTI definitions is to develop the useful guidelines for the clinical management and treatment options for patients with SSTIs (1,3). The annual frequency of visits to physicians offices for SSTIs have an increasing trend (4). The predominant pathogens associated with SSTIs in hospitalized patients include S. aureus (ranked first in all geographical regions), Pseudomonas aeruginosa, Escherichia coli and Enterococcus spp. (5). Methicillin-resistant Staphylococcus aureus (MRSA) causes many infections, but most frequently SSTIs, such as cutaneous abscesses, furuncles and cellulitis. Thus, the prevalence of these infections has increased dramatically (6). Risk factors for MRSA SSTIs include the presence of an abscess, previous MRSA colonization/ infection, antibiotic prescriptions within 8 weeks, diabetes mellitus and hospital admission within the preceding year (6). Extended-spectrum beta-lactamase (ESBL) production is one of the main mechanisms of resistance to beta-lactam antibiotics in Enterobacteriaceae, so the therapeutic choices in infections caused by such strains are limited (7,8). Most ESBLs belong to SHV and TEM family, but recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M β-lactamase) has been reported (8). In contrast to TEM or SHV-ESBLs, CTX-M β-lactamases are native ESBLs and are derived from the chromosomal β-lactamases of the genus Kluyvera (9). In many countries CTX-M β-lactamases are the most prevalent type of ESBLs (9,10). Plasmidmediated AmpC β-lactamases are derived from chromosomal ampc genes of the family Enterobacteriaceae. AmpC enzymes encoded by both chromosomal and plasmid genes are also evolving to hydrolyze broad-spectrum cephalosporins more efficiently (11). Since most skin and soft tissue infections in outpatient settings are treated with empiric antimicrobial therapy, it is very important to estimate the prevalence of causative agents associated with skin and soft tissue infections, as well as their antimicrobial resistance patterns and mechanisms (3-5). The aim of this study was to determine prevalence and molecular epidemiology of SSTIs caused by MRSA, ESBL- and plasmid-mediated AmpCproducing β-lactamase Gram-negative bacteria in the in- and outpatient settings in Zenica-Doboj Canton, Bosnia and Herzegovina (B&H). MATERIALS AND METHODS Setting, bacterial isolates and study design The Cantonal Hospital Zenica, B&H, is a 849- bed tertiary level hospital admitting about patients/year, with patient days, and covers a population of in Zenica-Doboj Canton, B&H. All consecutive, non-duplicate strains identified as MRSA and/or ESBL- or plasmid-mediated AmpC β-lactamase-producing Gram-negative bacteria obtained from SSTIs of the in- and outpatients during the period December 2009 May 2010 were analyzed. The SSTIs comprise surgical wound infections (SWIs) (postoperation and postraumatic wound infection) and other SSTIs (osstis) (including furuncles/abscesses, cellulitis, folliculitis) documented by the clinical provider/physician). Clinical and epidemiological data recorded for the patients involved in the study included: age, gender, occupation, place of residence at admission to the hospital (e.g. at home, other hospital, nursing home), contact with person(s) with history of hospitalization in the past 12 months, hospital department, antibiotic usage in the past 158

57 Uzunović et al. MRSA and ESBL skin and soft tissue infections four months, isolated causative agent (MRSA and/or ESBL or plasmid-mediated AmpC β-lactamase-producing Gram negative bacteria). An institutional review board approval had been obtained from the Ethics Committee in the Cantonal Hospital of Zenica prior to the initiation of the study, and all the participants read and signed informed consents about the purpose of the study (participation was voluntary and anonymous) as well. Identification of MRSA and susceptibility testing Staphylococcus aureus isolates were identified according to standard microbiological methods. The strains were tested for oxacillin and cefoxitin sensitivity/resistance by disk-diffusion method at Mueller-Hinton (MH) agar (Oxoid, Basingstoke, UK) (growth zone inhibition around 1 µg and 30 µg oxacillin and cefoxitin disk, respectively) in accordance with CLSI (Clinical Laboratory Standards) guidelines (12). All S. aureus isolates were analyzed for the presence of the S. aureus-specific fema gene as well as the MRSA-specific meca gene using a multiplex real-time PCR assay (13). The disc diffusion method using Mueller-Hinton agar (Oxoid, Besingstoke, UK) was used to test susceptibility to 11 antimicrobials (Oxoid, Basingstoke, UK): mupirocin, MUP (200 µg), imipenem, IPM (10 µg), erythromycin, ERY (15 µg), vancomycin, VAN (30 µg), gentamicin, GEN (10 µg), amikacin, AMK (30 µg), ciprofloxacin, CIP (5 µg), clindamycin, CLI (2 µg), trimethoprim/ sulfamethoxazole, SXT (25 µg), chloramphenicol, CHL (30 µg), and rifampicin, RIF (5 µg). The susceptibility testing results were interpreted according to CLSI (12). Staphylococcus aureus ATCC control strain was used for quality control. Multidrug resistance (MDR) was defined as resistance to three or more groups of antibiotics. Susceptibility testing of ESBL and AmpC producing bacteria The susceptibility testing to cefuroxime (CXM), ceftazidime (CAZ), ceftriaxone (CRO), cefotaxime (CTX), cefoxitin (FOX), tazobactam (TZP), cefepime (FEP), gentamicin (FEP), ciprofloxacin (CIP), and piperacillin (PIP) was performed by a twofold microdilution technique according to CLSI standard procedures (12). Susceptibility to imipenem (10 μg), meropenem (10 μg), tetracycline (30 μg), chloramphenicol (30 μg) and sulphametoxazole /trimethoprim (23.75 /1.25 μg) was performed by disk diffusion test (12). Phenotypic detection of ESBLs and plasmidmediated AmpC ß-lactamases A double-disk-synergy test (DDST) using the combination of amoxycillin/clavulanate with cefotaxime, ceftriaxone, ceftazidime, and cefepime was performed to detect the production of ESBLs. Distortion of the inhibition zones around cephalosporin and aztreonam disks towards central disk was considered as a positive result (14). Production of ESBLs was confirmed by CLSI combined disk test. Production of plasmid-mediated AmpC β-lactamase was tested in E. coli, Klebsiella spp. and P. mirabilis by combined disk test using 3-amino phenyl boronic acid. An overnight Mueller-Hinton (MH) broth culture of the strains was adjusted and swabbed on MH agar and disks of cefotaxime, ceftriaxone, ceftazidime and cefepime were placed on the surface of the agar plate. 10 µl of 3-amino phenyl boronic acid (20 mg/ ml) was dropped on the disks containing cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg) and cefepime (30 µg). Control plate contained disks of the same cephalosporins without phenyl boronic acid. Augmentation of the inhibition zones around cephalosporin disks for 5 mm in the presence of boronic acid was indicative for production of AmpC β-lactamases (15). Transfer of resistance determinants The transferability of cefotaxime resistance was tested by conjugation (broth mating method). Enterobacteriaceae were investigated for the transferability of their resistance determinants. Conjugation experiments were set up employing plasmid-free and rifampin-resistant E. coli A15 R - recipient strain (15). Transconjugants were selected on the combined plates containing cefotaxime (1 mg/l) and rifampicin (256 mg/l). The frequency of conjugation was expressed relatively to the number of donor cells. Typing of the spa locus of MRSA isolates Real-time amplification of the spa locus followed by sequencing was performed as des- 159

58 Medicinski Glasnik, Volume 12, Number 2, August 2015 cribed above (16). The spa types were clustered into spa CCs (clonal complexes) using the algorithm based upon repeat pattern (BURP) with the Ridom Staph Type, version 1.5, software package ( (17). The default settings recommended by the manufacturer were used. Since it has been shown that spa typing, together with the algorithm BURP, yields results consistent with typing results obtained by MLST (17-19), the associated CCs, as determined with MLST, were allocated through the Ridom SpaServer ( spaserver.ridom.de). Molecular characterization of ESBL and plasmidmediated AmpC ß-lactamases PCR was used to detect alleles encoding ESBL enzymes. Extended-spectrum β-lactamases were characterized at the molecular genetic level. The presence of bla TEM, bla SHV, bla CTX-M, ESBL genes was investigated by polymerase chain reaction (PCR) using primers and conditions as described previously (9,20,21). Template DNA was extracted by boiling method. PCR mix (50 µl) contained 25 µl of master mix (Roche), 20 µl of ultrapure water, 1 µl of each primer (10 pmol) and 3 µl of template DNA. Strains positive for CTX-M beta-lactamases were further tested by multiplex PCR with primers specific for CTX- M groups 1, 2, 8, 9 and 25 (22). Amplicons were column-purified (Quiagen DNA purification kit) and sequenced directly using ABI PRISM 377 Genetic Analyzer (Applied Biosystems). Sequences were analyzed using BioEdit v (Ibis Biosciences) program. Designation of bla genes based on identified mutations was done according to Bush and Jacoby (23). Primers IS26F (5 -GCG-GTA-AAT-CGT-GGA- GTG-AT-3) and IS26R (5 -ATT-CGG-CAA- GTT-TTT-GCT-GT-3 ) were used to amplify 400 bp fragment spanning the link between IS26 insertion sequence and bla CTX-M gene in CTX-M producing isolates (22). Primers ISEcp1L1 (CAGCTTTTATGACTCG) and ALA-5 (CCTAAATTCCACGTGTGT) were applied to amplify the ISEcpI insertion sequence (10). Multiplex PCR with primers specific for MOX, CMY, DHA, and FOX β-lactamases was used to detect plasmid-mediated Amp β-lactamases in E. coli, Klebsiella spp. and P. mirabilis strains resistant to cefoxitin and β-lactam/inhibitor combinations (11). Typing by pulsed-field gel electrophoresis (PFGE) of bacterial DNA Isolation of genomic DNA, its digestion with the XbaI restriction enzyme (Invitrogen) and PFGE of the resulting fragments was performed as described by Kaufman et al. (24,25). The electrophoresis was carried out with a CHEF-DRII apparatus (Bio-Rad Laboratories, Hercules, CA). The PFGE patterns were compared following the criteria of Tenover et al. (26) and analyzed by the GelComparII software (Applied Maths, St Martens, Belgium). The patterns obtained were compared by clustering methods (unweighted pair group methods with arithmetic averages) using the Dice coefficient. The optimization of 0.5% and position tolerance of 3% were applied. RESULTS During the period December 2009 May 2010, 33 hospitalized patients with SSTIs caused by MRSA or/and ESBL-producing Gram-negative bacteria were identified: 17 patients had infection caused by MRSA, 13 patients had infection caused by ESBL-producing Gram-negative bacteria, and three patients had co-infection with MRSA and ESBL-producers. MRSA infections Among 20 in-patients infected with MRSA, six (30%) had surgical wound infections (SWI) and 14 (60%) had osstis. Three patients with MRSA (two were spa-type t355) infection had co-infection with MSSA (one patient with osst at Dermatology, and two patients with SWI at surgery and orthopaedic department, respectively). Five (25%) in-patient and two (out of five) outpatient isolates were susceptible to all antibiotic tested, respectively. None of the isolates were multidrugresistant. Most in-patient isolates have shown gentamicin resistance phenotype, 13 (65.0%). In outpatient settings five SSTIs were noted, all were osstis; one was MRSA MLST CC152 (newborn). Among 14 osstis, all but one MRSA belonged to spa-clonal-complex (CC) 355/595 associated with MLST CC152. Among six MRSA isolated 160

59 Uzunović et al. MRSA and ESBL skin and soft tissue infections from SWIs, MLST CC152 was found in three cases (Table 1). Most in-patients with MRSA SSTIs were admitted to the hospital from home, with an exception of five patients who were transferred from other hospital or from nursing home (four MRSA belonged to MLST CC152) (Table 1). All hospitalized patients with SSTIs had contact with persons with positive history of hospitalization. A history of β-lactam antibiotics usage in combination with glycosides was positive in 16 patients (data not shown). Table 1. Characteristics of patients with MRSA infections and susceptibility /resistance to antibiotics Protocol No Gender Isolate origin Age HospItal (years) department Spa-type (Spa CC) (MLST CC) Residance before hospitalization Susceptibility/resistance to antimicrobial agents* IMP ERY VAN GEN AMK CIP CLI SXT CHL RA MUP 3196 F ossti 42 t355 Dermatology (355/595) (152) Home S S S R S S S NT NT S S M ossti 40 t355 Dermatology (355/595) (152) Home NT S S R NT S S S S S S 245 F SWI 54 ICU t355 Other (355/595) (152) hospital S S S R S S S NT NT S S M SWI 60 Internal t1855 (singleton) Home NT S S S NT S S S NT S S F SWI 45 Orthopedics t041 Other (002) (005) hospital NT S S S NT S S S NT S S 4357 F ossti (umbilicus) (355/595) (152) center t355 Health care <01 Pediatrics S S S S S S S NT NT S S 2236 M ossti 01 Pediatrics t355 Health care (355/595) (152) center S S S R S S S NT NT S S F ossti 01 Pediatrics t355 (355/595) (152) Home NT S S S NT S S R S S S 2822 F ossti 01 Pediatrics t355 (355/595) (152) Home S S S R S R S NT NT S S M ossti 01 Pediatrics t355 (355/595) (152) Home NT S S R S S S S S S 4189 M ossti <01 Pediatrics t355 (355/595) (152) Home S S S R S S S NT NT S S M ossti <01 Pediatrics t355 (355/595) (152) Home NT S S R NT S S S S S S 8723 M ossti <01 Pediatrics t355 (355/595) (152) Home S S S R S S S NT NT S S 5928 M ossti (umbilicus) (355/595) (152) t355 <01 Pediatrics Home S S S R S S S NT NT S S 9522 F ossti <01 Pediatrics t355 (355/595) (152) Home S S S R S S S NT NT S S F ossti 02 Pediatrics t595 (355/595) (152) Home NT S S R NT S S S S S S M ossti <01 Pediatrics t919 (008/024) (008) Home NT S S S NT S S S S S S F SWI 01 Surgery t355 (355/595) (152) Home NT S S R NT S S S S S S M SWI <01 Surgery t355 Health care NT (355/595) (152) center S S R NT S S S S S S 129/U M SWI Urology t7250 (singleton) Home S S S S S S S S S S 7559 M ossti 01 Outpatient t728 (015) (045) DM S S S S S S S S S S S F ossti (umbilicus) (355/595) (152) t Outpatient DM NT S S S NT S S S NT S S F ossti <01 Outpatient Not typeable DM NT R S R NT R S R S S S M ossti 29 Outpatient t451 (008/024) (008) DM NT R S R NT S S R S S S M ossti 01 Outpatient Not typeable NT S S R NT S S S S S S *IMP, imipenem (10 µg); ERY, erythromycin (15 µg); VAN, vancomycin (30 µg); GEN, gentamicin (10 µg), AMK, amikacin (30 µg); CIP, ciprofloxacin (5 µg); CLI, clindamycin (2 µg); SXT, trimethoprim/sulfamethoxazole (25 µg); CHL, chloramphenicol (30 µg); RIF,. rifampicin (5 µg); MUP, mupirocin (100 µg); Patients with MRSA and ESBL-producing Gram-negative bacteria coinfection Spa- CC, spa clonal complex; MLST CC, MLST clonal complex; SWI, surgical wound infection; ossti, other skin and soft tissue infection (other than SWI); NT, not tested; DM, data missing; 161

60 Medicinski Glasnik, Volume 12, Number 2, August 2015 Among 23 MRSA strains analyzed by PFGE, 15 (65.2%) belonged to the same clonal complex (Figure 1). ESBL infections Among 16 in-patients with infection caused by ESBL-producing Gram-negative bacteria two (12.5%) had SWI and 14 (87.5%) had ossti. Most ossti hospital infections were registered at pediatric department, nine (56.3%) (all were newborns). ESBL-producing Klebsiella spp. was the most frequently isolated Gram-negative bacteria, in 12 (75%) in-patients of which K. pneumonia was isolated in 11 cases. The total number of 18 outpatients were infected with ESBL-producing Gram-negative bacteria, of which 17 (94.4%) had SWI and one had os- STI. Two outpatients had co-infection with two Gram-negative bacteria (K. pneumoniae in both cases with K. pneumoniae and Pseudomonas aeruginosa, respectively). Klebsiella spp. was the most isolated, in seven (38.9%) cases. The four in-patients were transferred from another hospital. Eleven (61.1%) outpatients were 60 years of age (Table 2). Almost all in-patient ESBL-producing Klebsiella spp. isolates were resistant to gentamicin. Resistance to ciprofloxacin was noted in six Klebsiella spp. from in-patients, and in four outpatients. Two E. coli isolates have shown high-level resistance to almost all tested antibiotics. From one of these, MRSA was isolated too. All but one (Pseudomonas) isolates remained susceptible to carbapenems (Table 2). Eight strains transferred cefotaxime resistance to E. coli recipient strains with frequency ranging from 10-7 to Resistance to gentamicin, tetracycline, chloramphenicol and cotrimoxazole was cotransferred alongside with cefotaxime resistance in four strains (data not shown). Seven (out of 18, 38.9%) K. pneumoniae isolates (all were from the in-patients) yielded amplicons with primers specific for all three SHV, TEM-1 and CTX-M group 1 β-lactamases, one of which Figure 1. Dendogram showing the genetic relatedness of the 23 MRSA isolates. Two groups were identified by PFGE typing (A and B) by using 80% similarity. Group A consisted of two, group B of 15 isolates and they appeared to be clonally related S, singleton; ossti, other skin and soft tissue infections; SWI, surgical wound infections; NT, non-typeable; 162

61 Uzunović et al. MRSA and ESBL skin and soft tissue infections Table 2. Characteristics of patients with ß-lactamase producing Gram-negative bacteria causing skin and soft tissue infections and antibiotic susceptibility Protocol No gender Age (years) Isolate origin/ diagnosis Hospital department Causative agent isolated Residance before hospitalization Antibiotic (MIC in µg/ml)* Type of ß-lactamase AMX PIP TZP AMC CZ CXM CAZ CTX CRO FOX FEP GM CIP M 60 SWI outpatient E. coli Home NT NT > > TEM M 53 SWI outpatient E. coli Home 256 > >256 >256 > M 72 SSTI outpatient E.coli Home >256 > < , TEM, CTX-M F 71 SWI outpatient E.coli Home > TEM 8851 F 73 SWI ICU Enterobacter cloacae Other hospital 256 >256 8 >128 >128 > >128 8 > F 85 SWI outpatient Enterobacter cloacae Home >128 > AmpC F <01 ossti (umbilicus) Pediatrics Enterobacter cloacae Home 256 > >128 >128 >256 >256 >256 >256 > > SHV-1, TEM-1, CTX-M 15, OXA M 46 SWI outpatient Enterobacter cloacae Home >128 > TEM, CTX-M, OXA M 39 ossti Internal K. oxytoca Home >256 >256 > > SHV-1, TEM, CMY F 63 ossti (combustio, gangrena) Surgery K. pneumoniae Home 256 NT NT NT NT NT NT NT NT NT NT NT NT SHV-1, TEM, CTX-M M 01 ossti (umbilicus) Pediatrics K. pneumoniae Home 256 > >256 > > SHV-1, TEM-1, CTX-M 15, OXA F 01 ossti (umbilicus) Pediatrics K. pneumoniae Home 256 > > > > SHV F <01 Other SSTI (umbilicus) Pediatrics K. pneumoniae Other hospital >256 >256 > > SHV-1, TEM 5139 F <01 ossti (umbilicus) Pediatrics K. pneumoniae Home 256 > >256 >256 >256 >256 > ,5 64 SHV-1, TEM-1, CTX-M M 28 SWI outpatient K. pneumoniae Home > > SHV M 47 SWI outpatient K. pneumoniae Home >256 > SHV F <01 ossti (umbilicus) Pediatrics K. pneumoniae Home >256 >256 > >256 > SHV-1, TEM, CTX-M F 01 ossti (umbilicus) Pediatrics K. pneumoniae Home <0.12 >256 > > SHV F 74 ossti Internal K. pneumoniae Other hospital 256 NT NT 8 >256 NT NT NT NT NT NT NT NT SHV-1, TEM-1, CTX-M M 82 SWI outpatient K. pneumoniae Home >256 >256 <0,12 <0, <0, SHV F 76 SWI outpatient K. pneumoniae Home >256 > >256 >256 > >256 >128 SHV F <01 ossti (umbilicus) Pediatrics K. pneumoniae Home 256 > >256 >256 > > SHV-1, TEM-1, CTX-M M <01 Other SSTI (umbilicus) Pediatrics K. pneumoniae Home 256 > >256 >256 > >256 > SHV-1, CTX-M 1, OXA M 82 SWI outpatient K. pneumoniae Home >256 >256 > > SHV M 64 SWI (Coxarthrosis) Ortopedics/ traumatology K. pneumoniae Home 256 > > < <0,12 > SHV-1, TEM-1, CTX-M 15, OXA M 60 SWI outpatient K. pneumoniae Home 256 > >256 > <0,12 < <0, SHV-1, TEM M <01 ossti (umbilicus) outpatient K. pneumoniae Home 256 > SHV-1, CTX-M M 40 ossti Dermatology P. aeruginosa Home >256 >256 > > > VIM M 47 SWI outpatient P. aeruginosa Home NT NT > > F 45 ossti Orthopedics/ traumatology P. mirabilis Other hospital 256 > >256 >256 > > > M 28 SWI outpatient P. mirabilis Home >256 > >256 > >256 > M 68 SWI outpatient P. mirabilis Home 256 > >256 <0.12 <0,12 <0,12 1 < TEM F 65 SWI outpatient P. vulgaris Home 256 > >256 > > M 67 SWI outpatient Proteus vulgaris Home >256 > CTX-M, OXA1 *AMX, amoxycillin; PIP, piperacilin; TZB tazobactam; AMC, amoxycillin+clavulanic acid; CZ, cefazolin; CXM, cefuroxime; CAZ, ceftazidime; CRO, ceftriaxone; CTX, cefotaxime; FOX, cefoxitin; FEP, cefepime; GEN, gentamicin; CIP, ciprofloxacin; NT, non-tested; Type of ß-lactamase or AmpC, or positive phenotypic test for ESBL; Patients with MRSA-ESBL coinfection; Patient with two ESBL-producing strains coinfection; 163

62 Medicinski Glasnik, Volume 12, Number 2, August 2015 additionally produced plasmid-mediated AmpC β-lactamase. One of four E. coli (all from outpatients) coproduced both TEM and CTX-M β-lactamase. In one Pseudomonas aeruginosa isolate VIM β-lactamase was found. CTX-M beta-lactamases were most prevalent with 13 positive isolates (K. pneumoniae, E. coli, E. cloacae, Proteus vulgaris), and in five cases they were accompanied by both TEM-1 and OXA-1 betalactamase (Table 2). Insertion sequence IS26 was located upstream of bla CTX-M gene in two Enterobacter cloacae strains (data not shown). PFGE typing of K. pneumoniae using the 80% breakpoint for clonal relatedness revealed dominant cluster A which contained two subclusters: the clone A comprised 4 outpatient and the clone B two outpatient K. pneumoniae strains; three strains from pediatric and one from surgery hospital units were allocated in the dominant cluster A (Figure 2 A). Two E. coli strains were clonally related with 90% similarity of their banding patterns and assigned to cluster A, and one strain was singleton (Figure 2 B). MRSA/ESBL coinfection Among 17 and 16 in-patients with MRSA and ESBL infections, respectively, three patients were coinfected with both (Table 3). DISCUSSION S. aureus is a causative agent of large number of ambulatory healthcare visits for skin and soft tissue infections each year (6). The prevalence of MRSA-positive SSTIs has increasing trend, and up to 46-72% prevalence was noted (4,6,27,28). Of the SSTI cultures negative for MRSA, almost half are usually caused by methicillin-sensitive S. aureus (MSSA), 6% by gram-negative organisms, and 3% infections are polymicrobial (6). The Gram-negative ESBL producing bacteria were identified more commonly as a causative agent of post-surgical than other skin and soft tissue infections (7), and both Escherichia coli and Klebsiella spp. are among the most frequent Figure 2. Dendogram showing the genetic relatedness of the K. pneumoniae and Escherichia coli strains by PFGE typing. A) The clone A comprised four K. pneumoniae outpatient strains and the clone B comprised two outpatient strains using the 80% similarity; three strains from pediatric and one from surgery hospital units were allocated in dominant cluster A, which contained two subclusters; B) Two E. coli strains were clonally related with 90% similarity of their banding patterns and assigned to cluster A; one strain was singleton. SSTI, skin and soft tissue infection; SWI, surgical wound infection; 164

63 Uzunović et al. MRSA and ESBL skin and soft tissue infections Table 3. Patients with MRSA and ESBL-producing Gram-negative bacteria coinfection Patient Protocol No Age Hospital Isolate origin (years) department Causative agent Residance before hospitalization ATB used Patient SWI Orthopedics MRSA (spa-type t041; spa-cc 002; MLST CC5) Fluoroquinolones 45 Other hospital 2721 Other SSTI Orthopedics Proteus mirabilis (ESBL+) Glycosides Patient <01 SWI Other SSTI (umbilicus) Surgery Pediatrics MRSA (spa-type t355; spa-cc 355/595; MLST CC152) Enterobacter cloacae (TEM-1, CTX-M-15, SHV-1) Home Beta lactam+beta lactamase inhibitors Glycosides, penicillins Patient MRSA (spa-type t355; spa-cc 355/595; MLST 40 Other SSTI Dermatology CC152) Home Fluoroquinolones P. aeruginosa (VIM) * Patient with MRSA-ESBL coinfection; SWI, surgical wound infection; Other SSTI, skin and soft tissue infection other than SWI; enterobacteria producing ESBLs in these infections (7). Almost equal proportion of both SSTI caused by MRSA and MSSA was noted in this study (among 43 SSTIs identified during the study period caused by Staphylococcus spp., 39.5% were infected with MRSA, 41.9% with MSSA, 9.3% with S. epidermidis, and 9.3% of patients had co-infection with MRSA and MSSA) (Uzunović S, unpublished data). According to the results of this study ESBLproducing Gram-negative bacteria in outpatients were more frequently causative agents of SWIs, in contrast to the in-patients in which they more frequently caused other SSTIs. K. pneumoniae was dominant ESBL-producing pathogen of the in-patient SSTIs in this study, while the causative agents obtained from outpatient SSTIs were much more heterogeneous. A co-infection in SSTIs occurred frequently, mainly with Pseudomonas aeruginosa and MRSA (7), as demonstrated in this study. Moreover, two patients had co-infection with two ESBL-producing Gramnegative bacteria. Chronic infections, especially in patients previously treated with antibiotics, tend to be polymicrobial. Such mixed infections additionally complicate the antibiotic treatment and the outcome (29). Community-associated methicillin-resistant S. aureus (CA-MRSA) is the most common cause of SSTIs, especially in closed populations with frequent skin-to-skin contact (4,21). It is well known that skin infections occurred predominantly in children and young adults without risk factors, where family members can serve as a reservoir of CA-MRSA, so, the epidemic MRSA clone might be propagated in the community (30). As reported previously, an outbreak of CA- MRSA infections in a neonatal intensive care unit was initiated by a mother with CA-MRSA wound infection and mastitis (31). In the present study, it was not possible to differentiate between hospital-associated MRSA (HA-MRSA) and CA-MRSA. However, we found that all patients with MRSA infection in this study had contacts with persons recently hospitalized and who used antibiotics in the previous 4 months, which were identified as risk factors for HA-MRSA (6). Based on antimicrobial susceptibility testing, MRSA in this study was susceptible to a wide range of antibiotics, which is typical for CA-MRSA (27). From the genotyping results in this study, a MLST CC152 MRSA strains (Balkan clone) found in the in-patients (at multiple hospital departments), as well as in the outpatients, suggesting clonal spread by cross-transmission following introduction in the hospital (32,33), which was previously described in other reports (31,34). Indeed, PFGE results have shown that 80% of the MRSA strains belonged to one clone. Similar to MRSA, ESBLproducing K. pneumoniae were also clonally related indicating a common source. TEM- and SHV-type β-lactamases, mainly produced by K. pneumoniae, have spread throughout hospital settings, while CTX-M enzymes, mainly produced by E. coli, have become predominant in the community (35,36). ESBL-producing organisms are increasingly prevalent worldwide, and represent an emerging infectious threat, thus indicating that ESBL-producing organisms may be an emerging problem not only in hospital, but also in outpatient settings (35-37). The gut colonization of in-patients was identified as a risk factor for developing self and cross infections due to ESBL-producers, and further dissemination of ESBL-producing clones was a consequence of the transfer of patients between various units of the same hospital, but also between hospitals in the same countries, as well as across the borders (38). 165

64 Medicinski Glasnik, Volume 12, Number 2, August 2015 MRSA colonization of patients or their household members represents an important risk for subsequent MRSA infection (38). Nares and umbilicus were the two most common sites of MRSA colonization, and sampling of these two sites is necessary and might be adequate for surveillance cultures (24). Some authors suggest MRSA screening of family members, but others recommend the screening only for some categories of patients (40). Our study confirmed the importance of umbilicus as a possible site of the future infection and screening of mothers as possible sources of the future infection. The three most commonly described groups of SSTIs are other cellulitis or abscess, decubitus ulcer and post-operation wound infection (superficial infections), and they accounted for 76.5% of all hospitalized cases (41), which is confirmed in this study. 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66 Medicinski Glasnik, Volume 12, Number 2, August 2015 Meticilin-rezistentni S. aureus (MRSA) i gram-negativne bakterije koje proizvode ß-laktamaze proširenog spektra (ESBL) i plazmidom-posredovane AmpC ß-laktamaze kao uzročnici bolničkih i vanbolničkih infekcija kože i mekih tkiva Selma Uzunović 1, Branka Bedenić 2,3, Ana Budimir 3, Amir Ibrahimagić 1, Farah Kamberović 4, Zlatko Fiolić 5, Michelle I. A. Rijnders 6, Ellen E. Stobberingh 6 1 Služba za laboratorijsku dijagnostiku, Kantonalni zavod za javno zdravstvo Zeničko-dobojskog kantona, Zenica, Bosna i Hercegovina; 2 Medicinski fakultet, Sveučilište u Zagrebu, 3 Laboratorij za molekularnu mikrobiologiju, Klinički centar Zagreb; Hrvatska; 4 Mikrobiologija, Biotehniška fakulteta, Univerza v Ljubljani, Slovenia; 5 Kirurški odjel, Klinički centar Zagreb, Hrvatska; 6 Department of Medical Microbiology, The School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Center, Mastricht, The Netherlands ABSTRACT Cilj Istražiti meticilin-rezistentni S. aureus (MRSA) i gram-negativne bakterije koje proizvode ß-laktamaze proširenog spektra (ESBL) i plazmidom-posredovane AmpC ß-laktamaze kao uzročnike bolničkih i vanbolničkih infekcija kože i mekih tkiva (SSTI). Metode Osjetljivost na antibiotike određivana je disk-difuzijskom i mikrodilucijskom metodom u skladu s CLSI. MecA gen je određivan pomoću PCR-a, a genetička karakterizacija MRSA-e pomoću spatipizacije i BURP-a (algorithm based upon repeat patterns). Dvostruki sinergistički disk-test korišten je za probir ESBLs. bla ESBL aleli su detektirani pomoću PCR-a. Genetska srodnost između sojeva testirana je pomoću PFGE-a. Rezultati Kod bolničkih pacijenata izolirano je 17 MRSA, 13 ESBL-producirajućih gram-negativnih bakterija, kod tri pacijenta zabilježena je koinfekcija obje bakterije, a kod vanbolničkih pacijenata pet MRSA i 16 ESBL-producirajućih gram-negativnih bakterija. Klebsiella spp. izolirana je u 11 bolničkih i sedam vanbolničkih pacijenata. MLST CC152 bio je najprevalentniji MRSA. Kod sedam (38,9%) Klebsiella spp. detektirani su amplikoni s početnicama specifičnim za SHV, TEM-1 i CTX-M grupu 1 β-laktamaza. Osam (44,4%) sojeva K. pneumonia i 16 (64%) MRSA (bolničkih i vanbolničkih) pripadali su klonovima. Zaključak MRSA i ESBL-producirajuće gram-negativne bakterije koje uzrokuju infekcije kože i mekih tkiva vrijedne su pažnje zbog toga što usljed visokog morbiditeta i mortaliteta predstavljaju rizik za nastanak bolničkih infekcija. Ključne riječi: infekcije kirurških rada, CTX-M beta-laktamaze, MLST CC152, otpornost na antibiotike 168

67 ORIGINAL ARTICLE Emergence of extensive drug-resistant (XDR) Acinetobacter baumanniiin the Clinical Center University of Sarajevo, Bosnia and Herzegovina Amela Dedeić-Ljubović 1, Ðana Granov 1, Mirsada Hukić 2,3 1 Department of Clinical microbiology, Clinical Centre University of Sarajevo, 2 Department of Medical Science, Academy of Sciences and Arts of Bosnia and Herzegovina, 3 International Burch University; Sarajevo, Bosnia and Herzegovina ABSTRACT Aim Recently increased attention and interest for Acinetobacterbaumannii are the result of the occurrence of multidrug resistant (MDR), extensive drug resistant (XDR) and pandrug resistant (PDR) isolates around the world. The aim of this study was to examine the resistance of A. baumannii isolates to antimicrobials in Clinical Centre University of Sarajevo, Bosnia and Herzegovina. Methods Two hundred and fifty-seven A.baumannii isolates were collected between July 2011 and June 2012 in different wards and from different clinical samples. Multidrug resistant, XDR and PDR were defined according to international expert proposal for interim standard definitions for acquired resistance. Corresponding author: Amela Dedeić-Ljubović Department of Clinical microbiology, Clinical Centre University of Sarajevo Sarajevo, Bolnička 25, Bosnia and Herzegovina Phone: ; Fax: ; amela.ljubovic@hotmail.com Original submission: 23 February 2015; Revised submission: 21 April 2015; Accepted: 27 April doi: / Results A total of 257 A. baumannii isolates showed eleven different patterns of resistance, of which ten patterns corresponded to MDR and one corresponded to XDR (sensitive only to colistin). Multidrug resistant and XDR strains were the most common at Intensive Care Units and surgical departments. The largest numbers of isolates were found in wound swabs, blood and bronchial aspirate. Conclusion This is the first report of XDR A. baumannii in the 2000-bed Clinical Centre University of Sarajevo, Bosnia-Herzegovina. Although XDR strains have been detected, the resistance to colistin has not. The elevated prevalence of these strains indicates that local antibiotic prescription policies should be revised and infection prevention and control should be improved. Key words: antimicrobials, nosocomial infections, intensive care unit Med Glas (Zenica) 2015; 12(2):

68 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION Acinetobacter baumannii is a nonfermentative, gram-negative, nonmotile, oxidase-negative bacillus, whose natural reservoir still remains to be determined (1). Its ability to survive in a hospital milieu and its ability to persist for extended periods of time on surfaces makes it a frequent cause for healthcare-associated infections that include pneumonia, bacteremia, meningitis, urinary tract infection, wound infection and it has led to multiple outbreaks (2). Acinetobacter spp. can develop antibiotic resistance extremely rapidly what is in contrast to other clinical bacteria, which require greater time to acquire resistance, usually in response to therapeutic strategies (3). The emergence of antimicrobial-resistant Acinetobacter species is due both to the selective pressure exerted by the use of broad-spectrum antimicrobials and transmission of strains among patients, although the relative contributions of these mechanisms are not yet known (3). Antimicrobial resistance greatly limits the therapeutic options for patients who are infected with this organism, especially if isolates are resistant to the carbapenem class of antimicrobial agents (3). Definitions of multidrug-resistant Acinetobacter species vary and different terms like multidrug resistant (MDR), extensive drug resistant (XDR), and pandrug resistant (PDR) have been used to describe the extent of antimicrobial resistance among Acinetobacter spp. (4). A group of international experts came together through a joint initiative by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC), to create a standardized international terminology with which to describe acquired resistance profiles in all bacteria often responsible for healthcare-associated infections and prone to multidrug resistance(4). In the current review MDR Acinetobacter spp. is defined as the isolate resistant to at a least one agent in three or more antimicrobial categories, XDR Acinetobacter spp. as an isolate that is resistant to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories), and PDR Acinetobacter spp. as the XDR Acinetobacter spp. that is resistant to all agents in all antimicrobial categories (4). To date, there has been no record about extensive drug resistance (XDR) Acinetobacter baumanniiisolates in Bosnia and Herzegovina. Given the scarcity of data the aim of the present study was to examine the occurrence of resistant isolates of A. baumannii in the Clinical Centre University of Sarajevo. MATERIALS AND METHODS In this retrospective study which was conducted in Clinical Centre University of Sarajevo resistance to antimicrobials was observed in 257 Acinetobacter baumannii isolates in the period from July 2011 to June Isolates were detected from different clinical samples including urine, wound swab, blood, bronchial aspirate and other samples which were collected from patients admitted to various hospital wards. Identification of A. baumannii isolates was done on the basis of morphological, cultural and biochemical characteristics (5). In addition, automated VITEK 2 Compact system (biomérieux, Marcy l Étoile, France) was used to help with the confirmation of the A. baumannii and for antimicrobial susceptibility testing. Antimicrobial resistance profile was determined by disk-diffusion method for: amoxicillin/clavulanic acid, piperacillin/tazobactam, ceftriaxone, ceftazidime, cefepime, amikacin, gentamicin, tobramycin, imipenem, meropenem, ciprofloxacin, trimethroprim-sulphamethoxazole, colistin, minocycline. P.aeruginosa ATCC was used as quality control strain. Results were interpreted according EUCAST breakpoints (5). Multidrug resistant, XDR and PDR were defined according to international expert proposal for interim standard definitions for acquired resistance (4). RESULTS The survey contained 257 primo isolates of A.baumannii isolated from different samples and clinical departments in Clinical Centre University of Sarajevo from July 2011 to June Results of testing for antimicrobial resistance showed the presence of eleven (I-XI) different patterns of resistance (Table 1). The pattern VII belonged to XDR. Resistotype III (sensitive to colistin, tobramycin and minocycline) was the most frequent, 84 (33%) 170

69 Dedeić-Ljubović et al. Emergence of extensive drug resistant A. baumannii Table 1. Different patterns of resistance of A. baumannii isolates Pattern of resistance Antimicrobial agent* AN GA MEM IM FEP CAZ PIP AMC COL TOB TS CIP CRO MYN MDR/XDR I R S R R R R R R S R R R R S MDR II R R S S R R R R S S R R R S MDR III R R R R R R R R S S R R R S MDR IV R S R R R R R R S R R R R S MDR V R S R R R R R R S S R R R S MDR VI S R R R R R R R S S R R R S MDR VII R R R R R R R R S R R R R R XDR VIII S S R R R R R R S S R R R S MDR IX S S S S R R R R S R R R R S MDR X R R R R R R R R S S R R R S MDR XI S R R R R R R R S R R R R S MDR R, resistant; S, sensitive; *AN, amikacin; GA, gentamycin MEM, meropenem; IM, imipenem; FEP, cefepime CAZ, ceftazidime; PIP, piperacillin+tazobactam; AMC, amoxicillin+clavulanate; COL, colistin; TOB, tobramycin; TS, trimethroprim- sulfamethoxasole; CIP, ciprofloksacin; CRO, ceftriakson; MYN, minocycline strains, followed by the resistotype I (sensitive to colistin, gentamycin and minocycline), 59 (23%), resistotype VII (sensitive only to colistin), 37 (14.4%) and resistotype V(sensitive to colistin, gentamycin, tobramycin and minocycline), 21.8 (8.5%) isolates. From the total number of isolates of A.baumannii, 220 (85.6%) were MDR strains, while 37 (14.4%) were XDR strains (Figure 1). Multidrug resistant strains were most commonly obtained from Anesthesiology and Reanimation Unit, 39 (18.3%), Intensive Internal Therapy Unit, 33 (15.5%), and from Neurosurgery Intensive Care Unit (ICU), 22 (10.3%), while XDR strains were most commonly obtained from Plastic Surgery Unit, nine (24.3%), following with Anesthesiology and Reanimation Unit and Intensive Internal Therapy Unit, seven (18.9%) strains in each (Figure 3). Figure 1. Prevalence of different resistance patternsof 257 A. baumannii isolates The examination of time of the occurrence of XDR isolates showed that first strains were recorded in July 2011 (six isolates). The highest number of isolates were recorded in March 2012 (nine isolates). Multidrug resistant strains were isolated in the largest number in September 2011, May and April 2012, with 25 and 29 isolates, respectively (Figure 2). Figure 3. Multidrug resistant (MDR) and extensive drug resistant (XDR) Acinetobacter baumannii according to hospital departments ICU, Intesive Care Unit; IITU, Intensive internal Therapy Unit; ARU, Anestesiology and Reanimation Unit; FR Fiziatric and Rehabilitation Unit Analysis of isolates distribution according to the samples showed that the largest number of isolates were from wound swabs, 81 (31.5%), blood 48 (18.6%), and bronchial aspirate, 39 (15.1%) (Figure 4). Figure 2. Distribution 257 A. baumannii isolates in the period Figure 4. Multidrug resistant (MDR) and extensive drug resistant (XDR) A.baumannii according to the samples origin CVC, central venous catheter 171

70 Medicinski Glasnik, Volume 12, Number 2, August 2015 Monitoring of total resistance of isolates showed that there was no resistance to colistin, while resistance to minocycline was 14.4%. Resistance to aminoglycosides varied among isolates, to amikacin was the highest (87.9%), while the lowest was to tobramycin (45.2%). Resistance to other antimicrobials was higher than 90% (Figure 5). Figure 5. Overall resistance of A. baumannii to antimicrobials PIP, piperacillin+tazobactam; AMC, amoxicillin+clavulanate; TS, trimethroprim-sulfamethoxasole; S, Sensitive; R, Resistant DISCUSSION A. baumannii once considered opportunistic species and insignificant clinical pathogen today is one of the most important Gram-negative bacteria (6). It is responsible for various serious nosocomial infections particularly in intensive care units (7). A. baumannii can complicate the primary disease in severely ill patients and to increase the cost of the treatment (7). Studies has shown that the costs of treating patients infected with A. baumannii increased by an average of $ 60,916, and the hospital stay is longer by 13 days compared to patients who are not infected with this agent (7). Beside the ability of expressing resistance to many antibiotics, MDR, XDR and PDR strains have the ability for long-lasting survival in the inanimate surfaces, as well as the tendency for epidemic spread (8). Results of this study have shown 11 different resistance patterns among 257 A.baumannii isolates;85.6% isolates had 10 MDR patterns and 14.4% of isolates had one XDR pattern (sensitive only to colistin). Resistotypes III (sensitive only to colistin, tobramycin and minocycline) was the most frequent (33%). First XDR strains were recorded in July 2011 (6 isolates). In March isolates were recorded. In September 2011, May and April 2012 increased number of MDR strains were recorded with 25 and 29 of isolates, respectively. Multi-drug resistant and extensive resistant isolates of A. baumannii are increasingly reported all over the world, with the incidence range from 75% (Spain) (9), up to 100% (Italy, Greece, Turkey, Bulgaria) (10-12).In the neighboring countries there was also a high percentage of multi-drug resistant isolates of A. baumanniiand it ranges from 96.1% in Serbia to 100% in Croatia (13).Similar data were observed in distant parts of the world (USA, Taiwan, Iran, Jordan, China) where multidrug resistance in this microorganism ranged % (14-18). Study of Kuo et al. (19) showed that the prevalence of XDR A. baumannii increased significantly from 1.3% in 2002 to 41.0% in Tertiary Hospital in Central Part of Iran, also recorded elevated prevalence of XDR strains over a sixmonth period (20). A. baumannii isolates are common among patients in intensive care units and various surgical departments, primarily at the department for burns treatment. Incidence in theintensive care units ranges from 22% in Tunisia, 47-49% in China and Spain to 58.8% in Turkey, while in the departments of surgery from 30% to 62% (9, 21-23). Our data showed that A. baumannii isolates were commonly isolated among patients at the Anesthesiology and Reanimation Unit, Neurosurgery ICU, Intensive Internal Therapy Unit and Plastic Surgery Unit. It could be because patients in these sections were all in critical condition, they had extended hospitalization, lower immune defense, suffering from severe underlying diseases or frequent invasive procedures like tracheotomy. Risk factors for colonization or infection with multidrug-resistant Acinetobacter species include prolonged length of hospital stay, hospital size (over 500 beds), exposure to ICU, receipt of mechanical ventilation, colonization pressure, exposure to antimicrobial agents, recent surgery, invasive procedures, and underlying severity of illness (24-26). Wilkes et al. reported a recent outbreak of multidrug-resistant Acinetobacter infection, caused by environmental contamination (curtains, laryngoscope blades, patient lifting equipment, door handles, mops, and keyboards) (27). Medical equipment has been implicated, emphasizing the need for special attention to disinfection of shared items and extra 172

71 Dedeić-Ljubović et al. Emergence of extensive drug resistant A. baumannii caution with respiratory care and wound care procedures. One or more epidemic Acinetobacter clones often coexist with endemic strains making it difficult to detect and control transmission (3). In the present study, nearly 32% of A. baumannii recovered from clinical specimens were from wounds, blood, bronchial aspirate, and urine. A. baumannii causes a wide range of nosocomial infections. The most common infections are pneumonia in patients on artificial ventilation, bacteremia, urinary tract infections, wound infections, and less frequently meningitis. Very often these infections are associated with a high mortality rate (28). A. baumannii bacteremia caused a significant problem in hospitals worldwide. They are usually the result of pneumonia or catheter use and it is the most common cause of mortality in intensive care units (14). Studies from various parts of the world show different frequency of A. baumannii bacteremia. High prevalence of 92.5% is found in Korea (29). Slightly lower, but still high percentage of the A. baumannii bacteremia is found in studies from Brazil (78%) and Iran (60.5%) (30,14),while the incidence was significantly lower in China, Iran, Turkey and England and ranged from 15% to 52% (18,28,31,32). In our study over the period the isolates of A. baumannii exhibited high rate of resistance to all antibiotics tested. Resistance to carbapenems, cephalosporins (third and fourth generation), as well as to piperacillin-tazobactam, quinolones, and cotrimoxasole was over 95%. Among aminoglycosides resistance was lowest on tobramycin (below 50%).There was no resistance to colistin. The results of monitoring hospital infections caused by A. baumannii show that in recent years there has been a significant increase in its resistance to the most commonly used group of antibiotics (33). Except still good sensitivity to colistin A. baumannii is in high percentage (over 80%) resistant to cephalosporins (ceftazidime and cefepime), and the combination of piperacillin/tazobactam(34-36). The frequency of resistance to ciprofloxacin ranges from 85%, and even up to 100% (34-36). Among aminoglycosides data are different (34-36). The incidence of isolates resistant to gentamicin and amikacin ranges from 80-90%, while for tobramycin is below 75% (34-36). The percentage of carbapenem resistant varies from one country to another and ranges from 45% to over 90% (34-36). Development of multiresistance is contributed by previous usage of carbapenems, cephalosporins III generation, as well as fluoroquinolones and aminoglycosides (33). Certainly the most important is its ability to acquire resistance genes. Isolates of A. baumannii usually contain a set of genes encoding resistance to different groups of antibiotics at the same time (14). Of all multidrug resistant organisms (MDROs), carbapenemase-producing Acinetobacter spp. require special attention; this organism can be resistant to all currently available antimicrobial agents or remain susceptible only to older, potentially more toxic agents such as the polymyxins, leaving limited and suboptimal options for treatment (37). The problem of increasing resistance of A. baumannii is even more threatening when considering the very limited number of new antimicrobial agents that are in development (38,39). Management of Acinetobacter spp. infections is a great challenge for physicians and clinical microbiologists (40). Often colistin or tigecycline are the only available treatments for A. baumannii infections(41). Unfortunately, resistance to colistin has recently emerged in Europe. The European arm of the SENTRY surveillance program identified 2.7% of polymyxin B-resistant A. baumannii isolates collected during (41). In a recent surveillance study from Greece, among 100 A. baumannii strains derived from ICU patients, 3% were colistin resistant, whereas the minimum inhibitory concentration (MIC) levels of tigecycline ranged between μg/ml (42). A surveillance study performed in 34 centers across UK, during 2000, reported a 2% resistance rate to colistin among 443 A. baumannii tested, while tigecycline MICs ranged from < μg/ml (43).These data suggest that an antibiotic therapy should always be guided by in vitro susceptibility profile of the organism. The selective pressure caused by indiscriminate usage of broad-spectrum antibiotics in empirical therapy of hospital infections and environmental contamination is the main reason for such increased number of colonization and infection due to this highly resistant pathogen (20). In conclusion, A. baumannii are rapidly spreading with emergence of extended resistance to even newer antimicrobials. In this study we confirmed emergence of XDR A. baumannii strains in the most compromised patients in the ICUs, where all 173

72 Medicinski Glasnik, Volume 12, Number 2, August 2015 patients are in critical conditions, mostly intubated with long stay in hospital. Although we detected XDR strains, resistance to colistin wasnot detected. Carbapenem resistance was high, while somewhat lower resistance to aminoglycosides was recorded. The elevated prevalence of these strains indicates that local antibiotic prescription policies should be revised and infection control should be improved. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATIONS Competing interests: none to declare. REFERENCES 1. Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA. Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 2007; 51: Fournier PE, Richet H. The epidemiology and control of Acinetobacter baumannii in health care facilities. Clin Infect Dis2006; 42: Maragakis LL, Perl TM. 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73 Dedeić-Ljubović et al. Emergence of extensive drug resistant A. baumannii 23. Hammami S, Ghozzi R, Saidani M, Redjeb SB. Carbapenem-resistant Acinetobacter baumannii producing the carbapenemase OXA-23 in Tunisia. Tunis Med2011;89: Mireya UA, Martí PO, Xavier KV, Cristina LO, Miguel MM, Magda CM.Nosocomial infections in paediatric and neonatal intensive care units. J Infect 2007;54: Playford EG, Craig JC, Iredell JR. Carbapenem-resistant Acinetobacter baumannii in intensive care unit patients: risk factors for acquisition, infection and their consequences. J Hosp Infect 2007; 65: Nowak P, Paluchowska P, Budak A. Distribution of blaoxa genes among carbapenemresistant Acinetobacter baumannii nosocomial strains in Poland. New Microbiol2012;35: Wilks M, Wilson A, Warwick S,Price E, Kennedy D, Ely A, Millar ML. Control of an outbreak of multidrug-resistant Acinetobacter baumannii calcoaceticus colonization and infection in an intensive care unit (ICU) without closingthe ICU or placing patients in isolation. Infect Control Hosp Epidemiol2006; 27: Mostofi S, Mirnejad R, Masjedian F. Multi-drug resistance in Acinetobacter baumannii strains isolated from the clinical specimens of three hospitals in Tehran-Iran. Afr Jmicrobiol Res 2011;5: Choi SH, Choo EJ, Kwak YG, Kim MY, Jun JB, Kim MN, Kim NJ, Jeong JY, Kim YS, Woo JH. Clinical characteristics and outcomes of bacteremia caused by Acinetobacter species other than A. baumannii: comparison with A. baumannii bacteremia. J Infect Chemother 2006;12: Mostachio AK, van der Heidjen I, Rossi F, Levin AS, Costa SF. Multiplex PCR for rapid detection of genes encoding oxacillinases and metallo-βlactamases in carbapenem-resistant Acinetobacter spp. J Med Microbiol2009;58: Uskudar GA, Kilic A, Gozen AG, Bedir O, Basustaoglu A. Characterisation of carbapenemases in multidrug-resistant Acinetobacter baumannii isolates from intensive care units [abstract P637]. Proceedings of 21 st European Congress of ClinicalMicrobiology and Infectious Diseases (ECCMID)/27 th International Congress of Chemotherapy (ICC), Milan/Italy, May 7-10, Poster No 537. Clin Microbiol Infect 2011; 17 (Suppl. s4): S Wareham DW, Bean DC, Khanna P, Hennessy EM, Krahe D, Ely A, Millar M. Bloodstream infection due to Acinetobacter spp. epidemiology, risk factors and impact of multi-drug resistance. Eur J Clin Microbiol Infect Dis 2008;27: Souli M, Galani I, Giamarellou H. Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe. Euro Surveill 2008;13: Lin YC, Hsia KC, Chen YC, Sheng WH, Chang SC, Liao MH, Li SY. Genetic basis of multidrug resistance in Acinetobacter clinical isolates in Taiwan. Antimicrob Agents Chemother 2010;54: Goic-Barisic I, Bedenic B, Tonkic M, Katic S, Kalenic S, Punda-Polic V. First report of molecular characterization of carbapenem-resistant Acinetobacter baumannii in different intensive care units in University hospital Split, Croatia. J Chemother 2007;19: Bogiel T, Kwiecinska-Pirog J, Jachna-Sawicka K, Gospodarek E. Carbapenem-resistant Acinetobacter baumannii strains. Med Dosw Microbiol 2010;62: McGowan JJE. Resistance in nonfermenting gramnegative bacteria:multidrug resistance to the maximum. Am J Med 2006; 119 (suppl 1): European Centre for Disease Prevention and Control/European MedicinesAgency. ECDC/EMEA joint technical report: the bacterial challenge:time to react. European centre for disease prevention and control & European medicines agency, Stockholm, Sweden & London,United Kingdom, Boucher HW, Talbot GH, Bradley JS, Gilbert D, Rice LB, Scheld M,Edwards JE, Spelberg B,Bartlett J. Bad bugs, no drugs: noeskape! An update from the Infectious Diseases Society of America. Clin Infect Dis 2009; 48: Manchanda V, Sanchaita S & Singh NP.Multidrug Resistant Acinetobacter.J Glob Infect Dis 2010; 2: Gales AC, Jones RN, Sader HS. Global assessment of the antimicrobial activity of polymyxin B against clinical isolates of Gram-negative bacilli: report from the SENTRY antimicrobial surveillance programme. Clin Microbiol Infect2006;12: Souli M, Kontopidou FV, Koratzanis E, Antoniadou A, Giannitsioti E, Evangelopoulou P, Kannavaki S, Giamarellou H. In vitro activity of tigecycline against multiple-drug-resistant, including pan-resistant, gram-negative and gram-positive clinical isolates from Greek hospitals. Antimicrob Agents Chemother2006;50: Henwood CJ, Gatward T, Warner M, James D, Stockdale MW, Spence RP, Towner KJ, Livermore DM. Antibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936). J Antimicrob Chemother2002;49:

74 Medicinski Glasnik, Volume 12, Number 2, August 2015 Pojava ekstremno rezistentnih (XDR) sojeva Acinetobacter baumannii u Kliničkom centru Univerziteta u Sarajevu (Bosna i Hercegovina) Amela Dedeić-Ljubović 1, Ðana Granov 1, Mirsada Hukić 2,3 1 Klinički centar Univerziteta u Sarajevu, 2 Odjel za medicinske nauke, Akademija nauka i umjetnosti Bosne i Hercegovine, 3 Internacionalni Burch univerzitet; Sarajevo, Bosna i Hercegovina SAŽETAK Cilj Nedavno povećana pažnja i interesovanje za Acinetobacter baumannii rezultat je pojave multipli otpornih (MDR), ekstremno otpornih (XDR) i potpuno otpornih (PDR) sojeva širom svijeta. Cilj ovog istraživanja bio je ispitati otpornost A. baumannii izolata na antimikrobna sredstva u Kliničkom centru Univerziteta u Sarajevu (Bosna i Hercegovina). Metode Dvije stotine i pedeset sedam izolata A. baumannii prikupljeno je u periodu od jula do juna godine na različitim odjelima i iz različitih kliničkih uzoraka. Multiplootporni (MDR), XDR i PDR izolati definirani su u skladu s prijedlogom međunarodnih stručnjaka za donošenje privremene standardne definicije stečene rezistencije. Rezultati Kod 257 izolata A. baumannii otkriveno je jedanaest različitih tipova otpornosti od kojih deset odgovara MDR, a jedan XDR sojevima (osjetljiv samo na kolistin). Multipli otporni i XDR sojevi najčešće su izolirani u jedinicama intenzivne njege i hirurškim odjelima. Najveći broj izolata izolirano je iz brisa rane, krvi i aspirata bronha. Zaključak Ovom studijom su dokazani prvi izolati ekstremno otpornih sojeva (XDR) A. baumannii u Kliničkom centru Univerziteta u Sarajevu (Bosna i Hercegovina) koji raspolaže s bolesničkih kreveta. Iako su dokazani XDR sojevi, otpornost na kolistin nije otkrivena. Povišena prevalenca ovih sojeva pokazuje da se lokalna antibiotska politika treba revidirati, a kontrola i prevencija infekcija unaprijediti. Ključne riječi: antimikrobna sredstva, bolničke infekcije, jedinice intenzivne njege 176

75 ORIGINAL ARTICLE Brucellosis in children in Bosnia and Herzegovina in the period Sead Ahmetagić¹, Humera Porobić-Jahić¹, Nada Koluder 2, Lejla Čalkić 3, Snježana Mehanić 2, Eldira Hadžić 3, Nevzeta Ibrahimpašić 4, Svjetlana Grgić 5, Mirela Zirić 4, Jelena Bajić 6, Denis Žepić¹ 1 Clinic for Infectious Diseases, University Clinical Centre Tuzla, 2 Clinic for Infectious Diseases, University Clinical Centre Sarajevo, 3 Department for Infectious Diseases, Cantonal Hospital Zenica, 4 Department for Infectious Diseases, Cantonal Hospital Bihać, 5 Clinic for Infectious Diseases, Clinical Hospital Mostar, 6 Clinic for Infectious Diseases, Clinical Center Banja Luka; Bosnia and Herzegovina ABSTRACT Aim To analyse clinical, laboratory and epidemiological characteristics of brucellosis in children in Bosnia and Herzegovina. Methods The study included 246 children aged 0-18 years, who were hospitalized in Clinics and Departments for Infectious Diseases in Tuzla, Sarajevo, Banja Luka, Zenica and Bihać in the period , in whom the diagnosis of brucellosis was established based on anamnestic data, clinical features and positive results from blood culture and/or positive results from one of the serological tests. Corresponding author: Humera Porobić-Jahić Clinic for Infectious Diseases, University Clinical Center Tuzla Trnovac bb, Tuzla, Bosnia and Herzegovina Phone: ; Fax: ; humera.jahic@ukctuzla.ba Original submission: 03 March 2015; Revised submission: 21 April 2015; Accepted: 26 April doi: / Results In this period, a total of 2630 patients, 246 (9.35%) of whom were children, were treated from brucellosis at the Clinics and Departments in Bosnia and Herzegovina. In the majority of cases, the children were from rural parts of the country, 226 (91.87%);214 (87.04%) cases had direct contact with sick animals, sick family member or consumption of unpasteurized dairy products from farms where brucellosis had been already established. Male children predominated, 157 (63.82%). The most frequent clinical features in affected children were fever, 194 (78.86%) and joint pain, 158 (64.22%). The average duration of antimicrobial treatment was ± days. A total of 228 (92.68%) children were completely cured, while relapses occurred in 18 (7.32%) children. Conclusion Since brucellosis is an endemic disease in Bosnia and Herzegovina, it is important that physicians in their daily practice consider brucellosis and establish proper diagnosis and therapy in children with prolonged fever, arthralgia, leukopenia and positive epidemiological data, especially in rural parts of the country. Key words: clinical features, epidemiological characteristics, diagnosis, treatment Med Glas (Zenica) 2015; 12(2):

76 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION Brucellosis, also known as undulant fever, Mediterranean fever or Malta fever is a zoonosis, and the infection is almost invariably transmitted by direct or indirect contact with infected animals or their products (1,2). The major reservoirs of the disease include goats and sheep (Brucella melitensis), swine (Brucella suis), cattle (Brucella abortus) and dogs (Brucella canis) (3,4). It is an important infection of humans in many parts of the world such as Latin America, Southern Europe, Africa and Asia. In endemic, rural parts of the country the infection frequently affects all family members, including children regardless of their gender (5-8). The disease usually starts after consumption of unpasteurized milk and dairy products, and through contact with infected animals (9). Earlier, brucellosis in children was regarded a mild and rare disease, but today it is well known that brucellosis affects all age groups, especially in endemic countries (10-12). Brucellosis in endemic regions appears on average in 3% to 10% of children (4). Acute form of brucellosis is very frequent in children, with many nonspecific symptoms, and also clinical forms which affect musculoskeletal, gastrointestinal, genitourinary, hematopoetic, cardiovascular, respiratory and central nervous systems (11,12). Clinical manifestations in children are not significantly different from manifestations in adults (2,13,14). Bosnia and Herzegovina (B&H) was free from brucellosis from 1980 until Since then, the number of infected people in the country has rapidly increased, and infections have been recorded in almost the entire territory. Brucellosis reached its peak in 2008 for the observed period , with 778 patients recorded (15). Published papers from different centers in B&H indicated that brucellosis has become a public health problem in the country (16-18). The aim of this study was to analyze the clinical, laboratory and epidemiological characteristics of brucellosis in patients younger than 18 years, who were hospitalized in Clinics and Departments for Infectious Diseases in B&H, in the period from 2000 to PATIENTS AND METHODS The study included 246 children aged 0-18 years, in whom the diagnosis of brucellosis was established according to anamnestic data, epidemiological data, clinical features and in correlation with positive results of blood cultures and/or with one of the relevant serological tests. Patients were hospitalized in six Clinics and Departments for Infectious Diseases in B&H, Tuzla, Sarajevo, Mostar, Banja Luka, Zenica and Bihać in the period A retrospective analysis was conducted of the clinical, laboratory and epidemiological data on brucellosis, collected from medical records of patients younger than 18 years of age who were treated in Clinics and Departments for Infectious Diseases in B&H. Analyzed anamnestic data included: age, gender, place of living (urban or rural region), month of disease onset, contact with animals, consumption of raw milk or cheese, family history of brucellosis, animal farming on small rural households. Clinical symptoms and signs, laboratory findings, course and outcome of the disease were particularly analyzed. The diagnosis of brucellosis was established on the basis of anamnestic and epidemiological data, clinical features, and positive results from blood culture and/or one of the relevant serological tests (Rose-Bengal, CFT, Wright agglutination test, ELISA test). The study had been approved by the Research Ethics Committee of the University Clinical Centre Tuzla. RESULTS In the period , a total of 2630 patients with brucellosis, of whom 246 (9.35%) were children, were treated in six selected Infectious Disease Clinics and Departments in B&H (Table 1). In this period there were no declared outbreaks of brucellosis, because brucellosis is endemic in B&H and the frequency of cases is counted cumulatively, year by year. The largest number of infected children, 87 (out of 246, 35.36%) was registered in 2008, and the Table 1. Children with brucellosis in B&H in the period No (%) of patients with brucellosis City Total Children (0-18 years) Tuzla 196 (7.45) 12 (6.12) Zenica 885 (33.65) 104 (11.75) Sarajevo 672 (25.55) 58 (8.63) Bihać 589 (22.40) 54 (9.17) Mostar 64 (2.43) 8 (12.50) Banja Luka 224 (8.52) 10 (4.46) Total (9.35) 178

77 Ahmetagić et al. Brucellosis in children most cases were from Tuzla Canton, Una-Sana Canton and Central Bosnia Canton of B&H. The number of affected male children was 157 (63.82%), and female children 89 (36.18%). The average age of children was years ± The youngest child was a one- month old baby, and the oldest was 18 years old (Table 2). Table 2. Distribution of 246 children with brucellosis in B&H in the period according to age groups Age groups No (%) of patients (8.94) (15.45) (17.07) (28.86) (29.68) Total 246 (100) The majority of affected children came from rural regions, 226 (91.87%), of whom 214 (87.04%) had positive epidemiological data and/or confirmed direct contact with an infected animal, an infected family member or consumption of unpasteurized milk and dairy products from households in which brucellosis was already established. The consumption of raw milk or cheese was recorded in 26 (48.14%) patients. The most frequent clinical manifestations in affected children included fever in 194 (78.86%), joint pain in 158 (64.22%), malaise and tiredness in 125 (50.81%) and night sweating in 109 (44.30%) patients (Table 3). Table 3. Anamnestic data in 246 children (0-18 years) with brucellosis Anamnestic data No (%) of patients Contact with animal 197 (80.08) Sheep, goat or cow farming on a small rural household 116 (47.15) Shepherd 52 (21.13) Veterinarian 0 (00.00) Veterinarian technician 0 (00.00) Consumption of raw milk or cheese 155 (63.00) Unknown 33 (13.41) Family history of brucellosis 142 (57.72) Other clinical symptoms and signs in infected children are shown in Table 4. Elevated erythrocyte sedimentation rate (ESR) was recorded in 140 (56.81%) patients, increased values of CRP in 133 (54.06%), leucocytosis in twelve (04.87%), increased aspartate aminotransferase (AST) levels in 114 (46.34%), and increased alanine aminotransferase (ALT) levels in 40 (16.26%) patients. Decreased values of erythrocytes were registered in 58 (23.57%), thrombocytopenia in Table 4. Symptoms and signs in 246 children (0-18 years) with brucellosis Symptoms and signs No (%) of patients Fever 194 (78.86) Night sweating 109 (44.30) Headache 54 (21.95) Weakness 125 (50.81) Anorexia 56 (22.76) Weight loss 43 (17.48) Rash 11 (4.47) Cough 41 (16.66) Vomiting 27 (10.97) Diarrhea 18 (7.31) Stomach pain 42 (17.07) Frequent urination 6 (2.44) Dysuria 7 (2.84) Arthralgia 158 (64.22) One or more swollen joints 39 (15.85) Myalgia 55 (22.35) Hepatomegaly 27 (10.97) Splenomegaly 15 (6.09) Hepatosplenomegaly 58 (23.57) Testicular swelling 5 (2.03) Scrotal redness 5 (2.03) Scrotal pain 6 (2.44) Lymphadenitis 21 (8.53) 28 (11.38%) and decreased values of haemoglobin in 216 (87.80%) patients (Table 5). The diagnosis of brucellosis was established on the basis of positive blood cultures, Rose Bengal agglutination test, Elisa test and complement fixation test (CFT) (Table 5). Table 5. Laboratory findings in 246 pediatric patients (0-18 years) with brucellosis Laboratory finding Reference ranges No (%) of patients Erythrocyte sedimentation rate 20 mm/hours 140 (56.91) C-reactive protein mg/l 133 (54.06) Leukocytes x109/l 12 (4.87) Neutrophils % 5 (2.03) Lymphocytes % 151 (61.38) Monocytes % 9 (3.65) Erythrocytes % 58 (23.57) Hemoglobin g/l 216 (87.80) Thrombocytes x109/L 28 (11.38) Aspartate aminotransferase U/L 114 (46.34) Alanine aminotransferase U/L 40 (16.26) The number of patients with positive blood culture was 63 (25.61%). Brucella mellitensis was isolated from blood cultures in 20 (8.13%), Brucella abortus in 2 (0.81%), and Brucella species in 41 (16.66%) patients. The diagnosis of brucellosis was established only on the basis of serological tests in 183 (74.39%) patients. The number of patients with positive Rose Bengal (RB) test was 85 (34.55%), with positive ELISA test 35 (14.22%), and with positive RB and ELISA test 179

78 Medicinski Glasnik, Volume 12, Number 2, August (48.37%), while 7 patients had positive CFT and BAB reaction (rapid agglutination for brucella). Complications occurred in 64 (26.01%) patients as follows: monoarthritis in 15 (6.09%), polyarthritis in 16 (6.50%), synovitis in 8 (3.25%), spondylitis in 2 (0.81%), sacroileitis in 8 (3.25%), spondylodiscitis in 3 (1.22%), endocarditis in 1 (0.40%), pneumonia in 9 (3.66%), orchiepididymitis in 5 (2.03%) and splenic abscess in 5 (2.03%) patients. All hospitalized children received symptomatic antimicrobial therapy according to standard protocols. The average duration of treatment with antibiotics was ± days. The average hospital stay of infected children was ± days. Tetracycline in combination with rifampicin was used in 85 (34.56%), aminoglycosides in combination with tetracycline in 70 (28.46%), gentamycin + rifampicin in 7 (2.48%), gentamycin + trimetoprim-sulfamethoxasol (TMP-SMX) in 61 (24.79%) and triple therapy: gentamycin + rifampicin + TMP-SMX in 14 (5.69%) patients. Tetracyclines were used according to standard protocol in children older than 8 years of age. No fatal cases were registered or chronic forms of the disease. Relapses were registered in 18 (7.32%) cases. DISCUSSION Brucellosis was first diagnosed in B&H in 2000, and thereafter the number of patients constantly increased until 2008 (15), but from 2009, the number of patients has been decreasing. Until now the clinical, epidemiological and laboratory characteristics of brucellosis in children in B&H have not been systematically analyzed. Brucellosis in children was registered in 9.35% of the total number of cases of brucellosis in B&H in the observed period, which is very similar to the reported data on brucellosis in children in endemic regions like Mediterranean, Middle East and Latin America, were the incidence of brucella cases ranges from 3% to 10% (4). Bosilkovski et al (2010) reported that in the Republic of Macedonia in the period from 1998 to 2007, out of 550 registered cases of brucellosis, 86 (16%) were patients aged 0-14 years (19). Human brucellosis, as shown in our study, affected people living in rural regions, and was associated with the consumption of unpasteurized milk and dairy products. Similar data have also been reported by the majority of authors coming from other international endemic regions (9,14,20). Iranian authors have reported more frequent incidence of brucellosis in children coming from urban population (21). Shen from the USA (2014) has reported that controlling the disease in animals and humans significantly reduces the incidence of brucellosis in children in non-endemic countries (22). The fact that the majority of affected children (35.26%) were registered in 2008 is similar to reports from other endemic areas for brucellosis (19,23). Numerous studies have registered more frequent presence of brucellosis in boys than in girls (12,14,20). Data from our analysis that 58.54% of affected children were older than 10 years is also found in other reports from around the world (14,20,24). Brucellosis is a systemic disease that can involve any organ or organ system in the human body. The majority of our patients had clinical symptoms which were described by other authors as well (2,7,25). In our patients, the disease affected mostly bones and joints in the form of monoarthritis and polyarthritis, which has also been described by authors from Iran, Greece and B&H (6,21,26). Clinical manifestations in children were not significantly different from those in adults (1,4,27). In majority of published papers, the diagnosis of brucellosis was usually confirmed by positive blood cultures (24,25,28). Positive blood culture was recorded in 25.61% of our patients, which corresponds to data from around the world, where the percentage of positive blood culture ranges from 15% to 70% (5). Brucella melitensis was the most frequently isolated pathogen from blood in our country and in some other parts of the world as well (4,25). In all patients the disease was confirmed by positive blood cultures and/or serological Rose Bengal agglutination test, Elisa test, CFT. The clinical picture of brucellosis varies from very mild to severe. Antimicrobial treatment of six weeks or longer proved successful in 92.5 % of infected children (29). According to the World Health Organization (WHO) recommendations, the choice of antimicrobials for the treatment of brucellosis in children older than 8 years of age is the same as in adults. Since tetracyclines are 180

79 Ahmetagić et al. Brucellosis in children contraindicated in pregnant women and children younger than 8 years of age, alternative medicines are recommended in these groups of patients (4,30). In our study, all children were cured, and relapse occurred in 7.32% of cases. The most commonly used combination of antibiotics in children older than 8 years of age was doxycycline + rifampicin in 34.56% patients. To date many clinical studies have been published about the use of various antibiotics in the treatment of brucellosis in children (6,24,27). Impressive results were obtained from a prospective study of 1100 children with brucellosis, where the treatment scheme included a 3-week combination of TMP- SMX + streptomycin, gentamicin, or rifampicin (31). In another study in which a triple therapy was administered, relapses were not registered (24). In our study, a triple therapy was administered in a total of 23 (9.35 %) patients, and no relapses were recorded either in these patients. Our study suggests that in our country, which is considered endemic for brucellosis, it is necessary to harmonize the views and approach to the treatment of brucellosis patients as recommended by the WHO (4). In conclusion, brucellosis in children in Bosnia and Herzegovina is a disease that cannot be ignored. Considering its endemic nature, it is important that physicians in their daily practice consider brucellosis, and establish proper diagnosis and therapy in children with prolonged fever, arthralgia, leukopenia and positive epidemiological data, especially in rural parts of the country. Public health education is one of the most important methods for brucellosis prevention. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATIONS Conflict of interest: none to declare. REFERENCES 1. Jeren T. Brucella species. In: Begovac J, Božinović D, Lisić M, Baršić B, Schönwald S, Infektologija. 1st Ed. Zagreb: Profil, 2006; Young EJ. Brucella Species (Brucellosis). In: Long SS, Pickering LK, Prober CG. Principles and practice of Pediatric Infectious Diseases. New York: Churchill Livingstone Elsevier, 2008; 161: Abdussalam M, Fein DA. Brucellosis as a world problem. Dev Biol Stand 1976; 31: Corbel MJ. Brucellosis in humans and animals. Geneva: World Health Organization, 2006; Pappas G, Akritidis N, Bosilovski M, Tsianos E. Brucellosis. N Engl J Med 2005; 352: Giannakopoulos I, Nikolakopoulou NM, Eliopoulou M, Ellina A, Kolonitsiou F, Papanastasiou DA. Presentation of childhood Brucellosis in Western Greece. Jpn J Infect Dis 2006; 59: Bosilovski M, Krteva L, Dimzova M, Kondova I. Brucellosis in 418 patients from the Balkan Peninsula: exposure-related differences in clinical manifestations, laboratory test results, and therapy outcome. Int J Infect Dis 2007; 11: Dequi S, Donglou X, Jiming Y. Epidemiology and control of brucellosis in China. Vet Microbiol 2002; 90: Okur M, Erbey F, Bektaş MS, Kaya A, Doğan M, Acar MN, Uzun H. Retrospectiveclinical and laboratory evaluation of children with brucellosis. PediatrInt2012; 54: Sharda DC, Lubani M. A study of brucellosis in childhood. ClinPediatr 1986; 25: Feiz J, Sabbaghian H, Mirali M. Brucellosis due to B. melitensis in children. ClinPediatr 1978; 17: Mantur BG, Akki AS, Mangalgi SS, Patil SV, Gobbur RH, Peerapur BV. Childhood brucellosis: a microbiological, epidemiological and clinical study. J Trop Pediatr 2004; 50: Street L, Grant WW, Alva JD. Brucellosis in childhood. Pediatrics 1975; 55: Tsolia M, Drakonaki S, Messaritaki A, Farmakakis T, Kostaki M, Tsapra H, Karpathios T. Clinical features, complications and treatment outcome of childhoodbrucellosis in central Greece. J Infect 2002; 44: Obradović Z, Velić R. Epidemiological characteristics of brucellosis infederation of Bosnia and Herzegovina. Croat Med J 2010; 51: Ahmetagić S, Piljić D, Smriko-Nuhanović A, Ahmetagić A, Topalović B. Kliničke i epidemiološke karakteristike bruceloze u hospitaliziranih bolesnika. Infektol Glas 2008; 28: Tandir S, Sivić S, Toromanović S, Aličajić F. Epidemiology Features of Brucellosis at the Zenica-Doboj Canton Area in Period Med Arh 2008; 62: Krkić Dautović S, Hadžović Čengić M, Mehanić S, Ahmetagić S, Ibrahimpašić N, Hadžić E, Curić I, Derviškadić N, Bajić J, Bojanić J. Brucellosis-emerging zoonosis in Bosnia and Herzegovina. Int J Infect Dis 2010; 14: Bosilkovski M, Krteva L, Dimzova M, Vidinic I, Sopova Z, Spasovska K. Humanbrucellosis in Macedonia - 10 years of clinical experience in endemic region.croat Med J 2010; 51: Soleimani G. Evaluation of clinical findings and treatment of childhood Brucellosis in Zahedan. Iran J PediatrSoc 2010; 2:

80 Medicinski Glasnik, Volume 12, Number 2, August Zamani A, Kooraki S, Mohazab RA, Zamani N, Matloob R, Hayatbakhsh MR, Raeeskarami SR. Epidemiological and clinical features of Brucella arthritis in 24 children. Ann Saudi Med 2011; 31: Shen MW. Diagnostic and therapeutic challenges of childhood brucellosis in a nonendemic country. Pediatrics 2008; 121: Shahnaz A, Atoosa G, Abdollah K, Delara B, Nadere MK. Brucellosis in children: A disease with multiple features. JPediatr Infect Dis. 2007; El-Koumi MA, Afify M, Al-Zahrani SH. A prospective study of brucellosis in children: relative frequency of pancytopenia. Mediterr J Hematol Infect Dis 2013; 5:e Almuneef M, Memish ZA. Prevalence of Brucella antibodies after acute brucellosis. J Chemother 2003; 15: Mehanic S, Baljic R, Mulabdic V, Huric-Jusufi I, Pinjo F, Topalovic-Cetkovic J, Hadziosmanovic V. Osteoarticular manifestations of brucellosis. Med Arh 2012; 66: Ahmetagić S, Tihić N, Ahmetagić A, Čustović A, Smriko-Nuhanović A, Mehinović N, Porobić-Jahić H. Human Brucellosis in Tuzla Canton. Med Arh 2012; 66: Logan LK, Jacobs NM, McAuley JB, Weinstein RA, Anderson EJ. A multicenter retrospective study of childhood brucellosis in Chicago, Illinois from 1986 to2008. Int J Infect Dis 2011; 15: Al-Eissa YA, Kambal AM, Alrabeeah AA, Abdullah AM, al-jurayyan NA, al-jishinm. Osteoarticular brucellosis in children. Ann Rheum Dis 1990; 49: Grgić S, Nikolić J, Bradarić M, Skočibušić S. Epidemiološke i kliničke značajke bruceloze u djece. Infektol Glas 2012; 32: Lubani MM, Dudin KI, Sharda DC, Ndhar DS, Araj GF, Hafez HA, al-saleh QA, Helin I, Salhi MM. A multicenter therapeutic study of 1100 children withbrucellosis. Pediatr Infect Dis J 1989; 8:75-8. Bruceloza kod djece u Bosni i Hercegovini u periodu od do godine Sead Ahmetagić¹, Humera Porobić-Jahić¹, Nada Koluder 2, Lejla Čalkić 3, Snježana Mehanić 2, Eldira Hadžić 3, Nevzeta Ibrahimpašić 4, Svjetlana Grgić 5, Mirela Zirić 4, Jelena Bajić 6, Denis Žepić¹ 1 Klinika za infektivne bolesti, Univerzitetski klinički centar Tuzla, Tuzla, 2 Klinika za infektivne bolesti, Klinički centar Univerziteta u Sarajevu, Sarajevo, 3 Odjel za infektivne bolesti, Kantonalna bolnica Zenica, Zenica, 4 Odjel za infektivne bolesti, Kantonalna bolnica Bihać, Bihać, 5 Klinika za infektivne bolesti, Klinička bolnica Mostar, Mostar, 6 Klinika za infektivne bolesti, Klinički centar Banja Luka, Banja Luka; Bosna i Hercegovina SAŽETAK Cilj Ispitati kliničke, laboratorijske i epidemiološke karakteristike kod djece oboljele od bruceloze u Bosni i Hercegovini. Metode U ispitivanje je bilo uključeno 246 djece, u dobi do 18 godina, koja su bila hospitalizirana u klinikama i odjelima za infektivne bolesti u Tuzli, Sarajevu, Banja Luci, Zenici i Bihaću, u periodu od do godine, a kod kojih je bruceloza dijagnosticirana na osnovu anamnestičkih podataka, kliničke slike i pozitivnih rezultata hemokulture i/ili pozitivnih rezultata jednog od seroloških testova. Rezultati Od ukupno 2630 pacijenata liječenih od bruceloze u klinikama i odjelima u Bosni i Hercegovini, 246 (9,35%) su bila djeca. U većini slučajeva djeca su bila iz ruralnih područja, 226 (91,87%), a u 214 (87,04%) slučajeva imala su pozitivnu epidemiološku anamnezu o direktnom kontaktu s bolesnom životinjom, bolesnim članom porodice ili konzumacijom nepasteriziranih mliječnih proizvoda s farmi gdje je bruceloza već registrirana. Muška djeca su bila u većini, 157 (63,82%) slučajeva. Najčešći klinički simptom kod oboljele djece bila je povišena temperatura, 226 (78,86%), te bolovi u zglobovima, 158 (64,22%). Prosječno trajanje antimikrobne terapije bilo je 42,85 ± 10,67 dana. Ukupno 228 (92,68%) pacijenata bilo je potpuno izliječeno, dok se relaps pojavio kod 18 (7,32%) djece. Zaključak S obzirom da je bruceloza endemska bolest u Bosni i Hercegovini važno je da liječnici u svom svakodnevnom radu imaju na umu ovu bolest i postave pravu dijagnozu, te odgovarajući tretman djeci sa simptomima u vidu produžene temperature, artralgijama, leukopenijom i pozitivnom epidemiološkom dijagnozom, posebno u ruralnim dijelovima zemlje. Ključne riječi: klinički simptomi, epidemiološke karakteristike, dijagnoza, tretman 182

81 ORIGINAL ARTICLE Differences in newborn umbilical cord care Sanja Kanisek 1, Nada Prlić 2, Ivana Barać 2, Lorna Dubac Nemet 2 1 Community-Health Nursing Services, Community Health Centre Osijek, 2 University Study of Nursing, Faculty of Medicine, J. J. Strossmayer University of Osijek; Osijek, Croatia ABSTRACT Aim To investigate the frequency of different cord care practices as well recommendations to parents on cord care, along with the need to identify as well as reach the consensus on best cord care practices and other procedures in newborn care among health workers. Methods The study was conducted among 110 health care workers at the nursery departments in two general hospitals, six community-health nursing services and 16 pediatric practices in Eastern Croatia. The questionnaire created for this research has evaluated different cord care practices and recommendations to parents, a need to identify, as well as reach the consensus on best practices in cord care and other procedures in newborn care. Corresponding author: Sanja Kanisek Community-Health Nursing Services, Community Health Centre Osijek Park kralja P. Krešimira IV/6, Osijek, Croatia Phone: ; Fax: ; sanja.kanisek@gmail.com Original submission: Results Statistically significant differences have been found among respondent groups in three dry cord practices (p=0.000, p=0.002, and p=0.004, respectively) and three wet cord practices (p=0.000, p=0.001, and p=0.000, respectively). Significant differences were determined in three types of recommendations to parents about the care of dry cord (p=0.000, p=0.000, and p=0.002, respectively) and two recommendations for wet cord (p =0.000, p=0.000, respectively). The majority of respondents stressed the need for publishing guidelines on cord care, 104 (94.5%), and for other procedures in newborn care, 108 (98.2%). More than a half of respondents, 63 (57.3%), declared the need to reach a national agreement on guidelines for umbilical cord care. Conclusion Healthcare workers employ, as well as recommend, different umbilical cord care practices. It is necessary to prepare and reach a national agreement on written guidelines for umbilical cord care as well as for other procedures in newborn care. Key words: guidelines, health care workers 05 November 2014; Accepted: 02 February doi: / Med Glas (Zenica) 2015; 12(2):

82 Medicinski Glasnik, Volume 12, Number 2, August 2015 INTRODUCTION Umbilical cord stump and wound in newborns represent the point of entrance and development of systemic infections (1). This infection risk was the most frequent cause of changes in trends of newborn umbilical cord care practices over many years (2). The diversity in newborn cord care practices (3,4), suggests uncertainty about the most efficient care practice and was therefore the subject of many studies whose purpose was to demonstrate the most efficient newborn cord care regime (3). Based on the findings of many studies mostly from the developed countries, the World Health Organization (WHO) has published general guidelines on newborn umbilical cord care, recommending further, larger and longer-term studies (3). The principle of the WHO guideline is to keep the cord stump dry and clean; to apply antimicrobial agents in a hospital setting and in underdeveloped countries (3,5). Further studies acknowledge that there is not enough evidence to support the use of antimicrobial agents over keeping the cord stump dry and clean in developed countries, with the exception of preterm babies and newborns in Intensive Care Units (6). Croatian authors (1,7) recommend application of antimicrobial agents (70% isopropyl alcohol) in cord care, in contrast to WHO Guidelines which declared them not efficient enough in reduction of bacterial colonization of umbilical cord, as well as prolonging the cord stump healing (3). Differences in conducting cord care practices, which are often present within the same or different institutions (4,8,9), create the feeling of insecurity among healthcare workers (9). Healthcare workers inconsistence in conducting cord care practices as well as giving recommendations on cord care in a home setting (4,9,10), increases the already present maternal fear during the period of stump healing (9,10). Another important aspect, although not explicit, is the use of resources, whether it is the case of multiple visits of community-health nurse (11) or the amount of medical supplies used (11,12 ). Parents are additionally facing confusing diversity of recommendations on newborn cord care practices produced by healthcare workers such as midwives, nurses and pediatricians, based on beliefs rather than on scientific research findings (13). It is questionable whether there are any written guidelines on nursing interventions in newborn health care during stump healing phase, such as umbilical cord care, maintaining personal hygiene of a newborn or umbilical granuloma treatment with silver nitrate, in Croatian healthcare institutions. Moreover, it is arguable, whether the application methods of the mentioned procedures are evidence-based or simply relicts from the past, based on tradition, deeply rooted in the practice. The objectives of this study were to investigate the frequency of different cord care practices among healthcare workers, as well as their recommendations to parents on cord care during its healing related to healthcare worker s place of work, along with the need to identify as well as reach the consensus on best practices in cord care and other procedures in newborn care, (umbilical granuloma treatment with silver nitrate, personal hygiene). EXAMINEES AND METHODS Prior to the beginning of this study, the consent was issued by hospital managements and the Ethical Committee of the University Hospital Centre Osijek, General County Hospital Našice, Community Health Centers in Osijek, Našice, Valpovo, Donji iholjac, Beli Manastir and Đakovo. Participation in this study was completely voluntary and anonymous, and respondents were given both written and oral information on research. Written consent was obtained from all respondents. The study was conducted in April 2013 among healthcare workers involved in umbilical cord care in Eastern Croatia, Osijek-Baranja County. The study was conducted at the University Hospital Centre Osijek, Newborns Department, General County Hospital Našice, Newborns Department, community-health nursing services and pediatric primary care practices of the Community Health Centers in Osijek, Našice, Valpovo, Donji Miholjac, Beli Manastir and Đakovo. Anonymous questionnaire created for the purpose of this research was used as a research instrument, previously pretested and piloted on 15 health workers of different workplaces (hospital, community-health nursing services, pediatric primary care practice) and qualifications (nurse, bachelor of nursing, medical doctor). The questionnaire comprises socio-demographic data (age, gender, profession, workplace, years of service with current em- 184

83 Kanisek et al. Differences in umbilical cord care ployer), as well as questions on conduction of cord care practices and recommendations to parents on cord care during its healing ( dry umbilicus dry cord stump; wet umbilicus wet cord stump, wet wound), questions on availability of written guidelines and instructions on umbilical cord care for parents, questions on policies and rationales on maintaining hygiene in newborns during stump healing, questions on the necessity of creating guidelines on newborn cord care practices, reasons and levels, as well as questions on need to create and reach a mutual consent on other procedures in newborn care. Multiple response questions were introduced when questioning the necessity of creating guidelines on cord care practices. Respondents were asked to hand in a copy of written guidelines on umbilical cord practices and recommendations for parents, if available. There was a total of 35 open-ended and closed-ended questions. Before data processing, the respondents were divided into three groups according to their workplace (hospitals, community-health nursing services and pediatric primary care practices). Categorical data were presented in absolute and relative frequencies. Numerical data were described by arithmetic average and standard deviation. Differences between categorical variables were tested by χ² test and Fischer s Exact test. Distribution of numerical variables was tested by Kolmogorov-Smirnov test. The level of significance was set at α=0.05. RESULTS Out of 110 respondents who have participated in this study there were 22 (20%) hospital employees, 55 (50%) community-health nursing service employees and 33 (30%) pediatric primary care practice employees. In relation to their qualifications, there were 35 (32%) nurses, 57 (52%) bachelors of nursing and 18 (16%) medical doctors. The majority of the respondents were females, 108 (98.2%). The average age of the respondent was years, ranging from 20.0 to 64.0 years (SD; ). The average number of years of service with current employer was years, ranging from 1.0 to 45.0 years, (SD; ). Healthcare workers stated that they conducted a total of 11 dry cord care practices, whereby statistically significant differences were found according to workplace in three care practices: alcohol-free disinfectant, antibiotic powder and sterile gauze (p=0.000); alcohol-free disinfectant and sterile gauze (p=0.002) and antibiotic powder and sterile gauze (p=0.004). Healthcare workers stated that they conducted a total of six wet cord care practices, whereby statistically significant differences were also found according to workplace in three care practices: alcohol-free disinfectant, antibiotic powder and sterile gauze (p=0.000); 70% isopropyl alcohol, antibiotic powder and sterile gauze (p=0.001) and 3% hydrogen peroxide, saline solution, antibiotic powder and sterile gauze (p=0.000) (Table 1). Healthcare workers stated a total of nine types of recommendations for parents on dry umbilical cord care in a domestic setting. Statistically significant differences among the respondents of different professions were found in three recommended cord care practices: antibiotic Table 1. Dry (cord stump) and wet (cord stump/wound) umbilical cord care practices used by healthcare workers UC care practices Alcohol-free disinfectant, antibiotic powder, gauze 70% isopropyl alcohol, gauze 70% isopropyl alcohol, antibiotic powder, gauze Alcohol-free disinfectant, gauze 3% hydrogen peroxide, saline solution, antibiotic powder, gauze 3% hydrogen peroxide, antibiotic powder, gauze UC healing Number (%) of respondents phases H (n=22) C-HNS (n=55) PPCP (n=33) Total (n=110) p Dry 0 25 (45.6) 6 (18.2) 31 (28.2) Wet 0 27 (49.1) 8 (24.2) 35 (31.8) Dry 5 (22.7) 7 (12.7) 3 (9.1) 15 (13.7) Wet 4 (18.2) 4 (7.3) 2 (6.1) 10 (9.1) Dry 0 11 (20.0) 2 (6.0) 13 (11.8) Wet 0 19 (34.5) 4 (12.1) 23 (20.9) Dry 0 1 (1.8) 7 (21.2) 8 (7.3) Wet (9.1) 3 (2.7) Dry (6.1) 2 (1.8) Wet 18 (81.8) 4 (7.3) 10 (30.3) 32 (29.1) Dry 1 (4.5) 0 1 (3.0) 2 (1.8) Wet 0 1 (1.8) 6 (18.2) 7 (6.4) (45.6) 8 (14.5) 4 (12.1) 22 (20.0) Antibiotic powder, gauze Saline solution, antibiotic powder, gauze 5 (22.7) 1 (1.8) 2 (6.1) 8 (7.3) % hydrogen peroxide, saline solution, gauze Dry (9.1) 3 (2.7) Saline solution, gauze 0 1 (1.8) 2 (6.1) 3 (2.7) Gauze 1 (4.5) 1 (1.8) 1 (3) 3 (2.7) UC, umbilical cord; H, hospitals; C-HNS, community-health nursing services; PPCP, pediatric primary care practices 185

84 Medicinski Glasnik, Volume 12, Number 2, August 2015 Table 2. Recommendations for parents on dry (cord stump) and wet (cord stump/wound) newborn cord care practices in a domestic setting Recommendations on UC care practices in a domestic setting UC healing Number (%) of respondents phases H (n=22) C-HNS (n=55) PPCP (n=33) Total (n=110) p Alcohol-free disinfectant, antibiotic powder, gauze Dry 0 18 (32.7) 6 (18.2) 24 (21.8) Wet 0 31 (56.3) 11 (33.3) 42 (38.1) Antibiotic powder, gauze Dry 13 (59.1) 15 (27.3) 2 (6.1) 30 (27.3) Wet 0 4 (7.3) 2 (6.1) 6 (5.5) % isopropyl alcohol, antibiotic powder, gauze Dry 0 10 (18.2) 4 (12.1) 14 (12.8) Wet 0 15 (27.3) 5 (15.2) 20 (18.2) Alcohol-free disinfectant, gauze Dry 0 1 (1.8) 11 (33.3) 12 (10.9) Wet (9.1) 3 (2.7) % isopropyl alcohol, gauze Dry 5 (22.7) 4 (7.3) 2 (6.1) 11 (10.0) Wet 4 (18.2) 4 (7.3) 1 (3.0) 9 (8.2) % hydrogen peroxide, antibiotic powder, gauze Dry (15.1) 5 (4.5) Wet 1 (4.5) 1 (1.8) 6 (18.2) 8 (7.3) Gauze 0 5 (9.1) 1 (3.0) 6 (5.5) Saline solution, antibiotic powder, gauze Dry 4 (18.2) 1 (1.8) 0 5 (4.5) Pure water, drying, gauze 0 1 (1.8) 2 (6.1) 3 (2.7) % hydrogen peroxide, saline solution, antibiotic powder, gauze Wet 17 (77.3) 0 5 (15.1) 22 (20.0) UC, umbilical cord; H,hospitals; C-HNS, community-health nursing services; PPCP, pediatric primary care practices powder and sterile gauze (p=0.000); alcohol-freedisinfectant and sterile gauze (p=0.000) and 3% hydrogen peroxide, antibiotic powder and sterile gauze (p=0.002). Respondents listed a total of seven types of recommendations for parents on methods of wet newborn cord care in a domestic setting. Statistically significant differences among respondents of different professions were found in two recommended cord care practices: alcohol-free disinfectant, antibiotic powder and sterile gauze (p=0.000); and 3% hydrogen peroxide, saline solution, antibiotic powder and sterile gauze (p=0.000) (Table 2). For the majority of respondents the maintenance of personal hygiene in newborns during the umbilical stump healing time period (with the avoidance of wetting) had reduced the risk of infection, 88 (80.0%). Just17 (15.5%) of them considered that newborn cord care practices had no influence on risk of developing an umbilical cord infection, whereas five respondents (4.5%) were not familiar with it (p=0.870). As the most frequent method of and/or recommendation on personal hygiene maintenance in newborns during umbilical cord stump healing the healthcare workers highlighted the method of cleaning the baby on the changing mat with avoidance of wetting, 50 (45.5%), whereby statistically significant difference was found among groups (p=0.000) (Table 3). The availability of written guidelines on newborn umbilical cord care for healthcare workers at healthcare institutional level was acknowledged by only a small number of hospital respondents, 17 (15.5%). The majority of respondents, 89 (80.9%) stated that no written guidelines were available, just a verbal agreement, whereby four respondents (3.6%) were not familiar with the existence of written guidelines at institutional level. Statistically significant difference was found among groups of respondents (p=0.000). Only a small number of respondents, nine of them (8.2%), confirmed the existence of written recommendations for parents at institutional level. The majority of respondents, 99 (90%), stated the lack of written recommendations for parents, yet confirmed verbal instructions. In contrast, two respondents (1.8%) were not familiar with the existence of such recommendations. In view of that, statistically significant difference was also found among groups of respondents (p=0.000). Table 3. Methods of and/or healthcare workers recommendations on personal hygiene maintenance in newborns during umbilical cord stump healing Methods and/or recommendations While cleaning the baby on a changing mat avoid wetting the UC stump While washing the baby in a bath tub avoid wetting the UC stump While washing the baby under running water avoid wetting the UC stump While washing the baby in a bath tub the UC stump may get wet Number (%) of respondents H C-HNS PPCP Total (n=22) (n=55) (n=33) (n=110) 0 8 (36.4) 14 (63.6) 30 (54.5) 25 (45.5) (60.6) 11 (33.3) 1 (3.0) 1 (3.0) 50 (45.5) 44 (40.0) 15 (13.6) 1 (0.9) p UC, umbilical cord; H,hospitals; C-HNS, community-health nursing services; PPCP, pediatric primary care practices 186

85 Kanisek et al. Differences in umbilical cord care The majority of respondents, 104 (94.5%), agreed that there was a necessity to create written guidelines on newborn umbilical cord care, whereby six of them (5.5%) were of different opinion (p=0.004). As the most frequent reason for creating written guidelines on newborn umbilical cord care the respondents, 87 (76.4%), reported decrease and/ or elimination of parental confusion regarding differences in umbilical cord care practices among healthcare workers of different healthcare institutions, (p=0.668) (Table 4). Table 4. The reasons for the necessity of creating written guidelines on newborn cord care practices Number (%) of respondents Reasons H C-HNS PPCP Total p (n=22) (n=55) (n=33) (n=110) It would decrease and/or eliminate parental confusion regarding differences in UC care practices among healthcare workers of different healthcare institutions It would increase the sense of confidence and job satisfaction of healthcare workers (when I know precisely what to do/ how to do it) It would decrease and/or eliminate healthcare workers confusion regarding differences in UC care practices It would reduce the risk of newborn UC stump infection It would rationalize the resources used during UC stump healing (number of community-health nurse visits, medical supplies for UC care) 16 (72.7) 7 (31.8) 13 (59.1) 13 (59.1) 6 (27.3) 41 (74.5) 33 (60.0) 27 (49.1) 18 (32.7) 16 (29.1) 27 (81.8) 26 (78.8) 22 (66.7) 18 (54.5) 16 (48.5) 84 (76.4) 66 (60.0) 62 (56.4) 49 (44.5) 38 (34.5) UC, umbilical cord; H,hospitals; C-HNS, community-health nursing services; PPCP, pediatric primary care practices More than half of the respondents, 63 (57.3%), declared that the above-mentioned guidelines should be coordinated at the national level, (p=0.000) (Table 5). Table 5. The necessary level of coordination concerning the written guidelines on newborn umbilical cord care Level National level Local level (H with satellite C-HNS and PPCP) Level of particular healthcare institution Number (%) of respondents H C-HNS PPCP Total (n=22) (n=55) (n=33) (n=110) 3 (13.6) 18 (81.8) 1 (4.5) 31 (56.4) 24 (43.6) 0 29 (87.9) 3 (9.1) 1 (3.0) p 63 (57.3) (40.9) (1.8) H,hospitals; C-HNS, community-health nursing services; PPCP, pediatric primary care practices The majority of respondents, 108 of them (98.2%), declared that there was a necessity of creating and coordinating the guidelines for other newborn care practices (p=0.718). DISCUSSION The limitation of the study is that it was done only in one area of the Republic of Croatia. However, this kind of research in this area of nursing practice has no one tested so far and this paper is a good indicator of diversity among health professionals involved in the care of newborns navel. Healthcare workers involved in conducting umbilical cord care practices in Osijek-Baranja County, employ and recommend more different newborn cord care practices, depending on the healing phase. Recommendations to parents on cord care practices are not in line with the WHO guidelines on umbilical cord care in a non-hospital setting, which recommend keeping the cord stump/wound dry and clean, without application of any topical agents (3,5). The majority of our respondents conduct and/ or recommend maintaining of newborn personal hygiene without wetting the umbilical cord stump/wound because they consider it would reduce risk of cord stump/wound infection, which is not in line with the research results that contradict that point of view (5, 13-16). The availability of written guidelines for healthcare workers on newborn umbilical cord care at the institutional level is unanimously confirmed only by a small number of respondents coming from one and the same hospital, by enclosing a copy of their written guidelines. The results are similar to the research conducted in Scotland where the larger hospital departments share a tendency of having written guidelines (9). Ireland et al. stress that the non-existence of guidelines for abovementioned practices is a disappointing fact in the age of evidence-based practice (9). The research results show that almost half of the mothers after being discharged from the hospital are not able to recollect on verbal instructions on umbilical cord care practices in a domestic setting, i.e. only a small number of mothers claim that they have never got the aforementioned recommendations (10). The availability of written recommendations for parents on umbilical cord care in a domestic setting is confirmed by only a 187

86 Medicinski Glasnik, Volume 12, Number 2, August 2015 small number of respondents in this research, i.e. respondents coming from one pediatric primary care practice and only a half of the employees of the above mentioned hospital. However, the availability of written recommendations for parents at the hospital level is disputable for many reasons: aside from the fact that the copy of the recommendations was not enclosed, the question remains how it is possible that more than half of those respondents had no knowledge of the existence of the aforementioned recommendations. The listed results are the same as the ones obtained in a research conducted in Scotland, in which also a significant number of respondents from the same department claim existence/nonexistence of written recommendations (9). As the most frequent reason for creating written guidelines on newborn cord care practices, the respondents highlighted the decrease and/or elimination of parental confusion regarding differences in umbilical cord care practices among healthcare workers. The obtained results confirm the research results of Ford and Ritchie, who stress the fact that different recommendations on cord care practices presented by healthcare workers impose an additional source of parental concern (10). The majority of respondents in this research agree that there is a necessity for creating written guidelines on umbilical cord care and other newborn care practices (umbilical granuloma treatment with silver nitrate, methods and frequency of bathing, as well as the use of personal hygiene products), whereby more than a half of respondents believe that guidelines on umbilical cord care should be coordinated at the national level, which is in accordance with the research conducted in Scotland (9). With the availability of evidence-based clinical guidelines providing specific recommendations for nursing practice and consequently multiple benefits for patient and healthcare workers, it is to be expected that nursing based on experience, tradition or autonomy of authority is far behind us. However, the presented research results show that our nursing practice still lives in the past. Among healthcare workers involved in conducting umbilical cord care practices in Osijek-Baranja County there are statistically significant differences in methods of cord care conduction and recommendations to parents on cord care practices during stump/wound healing, in relation to the workplace (hospitals, community-health nursing service, pediatric primary care practices). The results emphasize the necessity of creating written guidelines on umbilical cord care and other newborn care practices, whereby the guidelines on umbilical cord care must be coordinated at the national level. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. REFERENCES 1. Mardešić D. Pedijatrija. Zagreb: Školskaknjiga, McConell TP, Lee CW, Couillard M, Sherrill WW. Trends in umbilical cord care: scientific evidence for practice. Newborn Infant Nurs Rev 2004; 4: World Health Organization. Care of the umbilical cord: a review of the evidence. rht/documents/msm98-4/msm-98-4.htm (07 April 2013). 4. Block SL. Stumped by the newborn umbilical cord. Pediatr Ann 2012; 41: World Health Organization Pregnancy, childbirth, postpartum and newborn care: a guide for essential practice. (07April 2013) 6. Zupan J, Garner P, Omari AAA. Topical umbilical cord care at birth. Cochrane Database Syst Rev 2004; 3:CD Malčić I, Ilić R. Pedijatrija sa zdravstvenom njegom. Zagreb: Školska knjiga, Guala A, Guarino R, Zaffaroni M, Martano C, Fabris C, Pastore G, Bona G, Neonatal Piedmont Group. The impact of national and international guidelines on newborn care in the nurseries of Piedmont and Aosta Valley, Italy. BMC Pediatr 2005; 5: Ireland J, Rennie AM, Hundley V, Fitzmaurice A, Graham W. Cord-care practice in Scotland. Midwifery 2000; 16: Ford LA, Ritchie JA. Maternal perceptions of newborn umbilical cord treatmens and healing. JO- GNN 1999; 28: Mugford M, Somchiwong M, Waterhouse IL. Treatment of umbilical cords: a randomised trial to assess the effect of treatment methods on the work of midwives. Midwifery 1986; 2: Mahrous ES, Darwish MM, Dabash SA, Marie I, Abdelwahab SF. Topical application of human milk reduces umbilical cord separation time and bacterial colonization compared to ethanol in newborns. Transl Biomed 2012; 3:1. 188

87 Kanisek et al. Differences in umbilical cord care 13. Blume-Peytavi U, Hauser M, Stamatas GN, Pathirana D, Bartels NG. Skin care practices for newborns and infants: review of the clinical evidence for best practice. Pediatr Dermatol 2012; 29: Bryanton J, Walsh D, Barrett M, Gaudet D. Tub bathing versus traditional sponge bathing for the newborn. JOGNN 2004; 33: Henningsson A, Nystrom B, Tunell R. Bathing or washing after birth? Lancet 1981; 2: Hylen AM, Karlsson E, Svanberg L, Walder M. Hygiene for the newborn: To bath or to wash? J Hyg 1983; 91: Razlike u njezi pupka novorođenčadi Sanja Kanisek 1, Nada Prlić 2, Ivana Barać 2, Lorna Dubac Nemet 2 1 Patronažna služba, Dom zdravlja Osijek, 2 Studij sestrinstva, Medicinski fakultet, Sveučilište Josipa Jurja Strossmayera u Osijeku; Osijek, Hrvatska SAŽETAK Cilj Ispitati pojavnost različitih načina provedbe i preporuka o njezi pupka te potrebu oblikovanja i usuglašavanja smjernica o njezi pupka i drugih postupaka u njezi novorođenčadi. Metode U istraživanju je sudjelovalo 110 zdravstvenih djelatnika na odjelima za novorođenčad u dvije opće bolnice, 6 patronažnih službi i 16 pedijatrijskih ordinacija u istočnoj Hrvatskoj. Upitnikom koji je kreiran za potrebe ovog istraživanja ispitivana je različitost načina provedbe i preporuka roditeljima o njezi pupka, te potreba oblikovanja i usuglašavanja smjernica o njezi pupka i drugih postupaka u njezi novorođenčadi. Rezultati Utvrđene su statistički značajne razlike među skupinama ispitanika u tri načina njege suhog (p=0.000, p=0.002 i p=0.004) i tri načina njege vlažnog pupka (p=0.000, p=0.001 i p=0.000). Također su utvrđene statistički značajne razlike u tri vrste preporuke roditeljima o njezi suhog (p=0.000, p=0.000, i p=0.002) i dvije vrste preporuke o načinu njege vlažnog pupka (p=0.000, p=0.000). Većina ispitanika, 104 (94.5%), suglasna je u potrebi oblikovanja pisanih smjernica o njezi pupka, kao i drugih postupaka u njezi novorođenčadi, 108 (98.2%). Više od polovice ispitanika, 63 (57.3%), izjasnilo se kako je smjernice o njezi pupka potrebno usuglasiti na nacionalnoj razini. Zaključak Zdravstveni djelatnici u istočnoj Hrvatskoj na različite načine provode i preporučuju provedbu njege pupka novorođenčadi. Potrebno je oblikovati i usuglasiti smjernice o njezi pupka na nacionalnoj razini, kao i smjernice za druge postupke u njezi novorođenčadi. Ključne riječi: smjernice, zdravstveni djelatnici 189

88 ORIGINAL ARTICLE Effects of perioperative statin treatment on postoperative atrial fibrillation and cardiac mortality in patients undergoing coronary artery bypass grafting: a propensity score analysis Ayşegül Kunt 1, Sedat Özcan 2, Aslihan Küçüker 3, Dolunay Odabaşi 1, Alper Sami Kunt 1 1 Department of Cardiovascular Surgery, School of Medicine, 100.Yil University, Van, 2 Department of Cardiovascular Surgery, School of Medicine, 18 Mart University, Çanakkale, 3 Department of Cardiac Surgery, Ataturk Education and Research Hospital, Ankara; Turkey ABSTRACT Aim To evaluate the effect of perioperative statin treatment on postoperative atrial fibrillation and cardiac mortality in patients undergoing coronary artery bypass grafting. Methods A total of 1890 patients who underwent isolated coronary artery bypass were analyzed retrospectively, of which 425 patients (22.4%) older than 70 were included in the study. The demographic properties, preoperative, operative and postoperative data and other medications of these patients were recorded. Continuous preoperative and postoperative atorvastatin therapy were received by 124 (29.17%) patients; 301 (70.82%) patients were matched to a control group (no-statin group). The two groups were matched by propensity score analysis in terms of atrial fibrillation development and cardiac mortality. Corresponding author: Sedat Özcan Department of Cardiovascular Surgery, School of Medicine, Canakkale 18 Mart University Çanakkale, Turkey Phone: Fax: sedatozcan78@hotmail.com Original submission: 20 January 2015; Revised submission: 13 March 2015; Accepted: 16 March doi: / Results Medical history, medical treatment, cardiovascular history, and operative characteristics demonstrated significant heterogeneity in both groups. Postoperative atrial fibrillation was similar in both groups. Before propensity score matching, the percentages of patients in postoperative atrial fibrillation with respect to Atorvastain-group and No-statin-group were and 10.3 respectively; however, those were and after matching. In a multivariate regression analysis, five-vessel bypass (odds ratio OR, 2.354; 95% confidence interval CI, 0.99 to 5.57) was an independent predictor of postoperative atrial fibrillation in patients undergoing coronary artery bypass grafting. In-hospital mortality was higher in the Atorvastatin-group compared with the No-statingroup: 124 (8.9%) versus 301 (3.7%), respectively; p=0.027). Conclusion Perioperative atorvastatin treatment is not found to be associated with reduced postoperative atrial fibrillation and cardiac mortality in patients undergoing isolated coronary artery bypass grafting above the age of seventy years. Key words: preoperative statin therapy, coronary artery bypass grafting, postoperative atrial fibrillation, cardiac mortality, propensity score analysis Med Glas (Zenica) 2015; 12(2):

89 Kunt et al. Preoperative statin treatment INTRODUCTION Atrial fibrillation (AF) occurs in 16% to 33% of patients undergoing coronary artery bypass grafting (CABG) (1,2). Patient age is one of the most important risk factors postoperative AF, more than 50% for patients older than 80 years undergoing CABG [3]. Moreover, a variety of pharmacological agents (4, 5) are thus commonly used to prevent AF after CABG. It is suggested that statins are associated with reduced risk of postoperative atrial fibrillation (6). In Atorvastatin for Reduction of Myocardial Dysrhythmia After Cardiac Surgery (ARMYDA-3 ) trial, preoperative atorvastatin treatment 7 days before CABG resulted in 61% decrease for the new-onset atrial fibrillation compared with placebo (35% vs 57%, respectively; p=0.003) (7). Postoperative AF increases hospital mortality (1, 8). Almassi and his colleagues first reported that 6-month survival after cardiac surgery decreased in patients affected by postoperative AF compared with patients without it (9.4% vs 4.2%, respectively) (9). We retrospectively evaluated whether perioperative atorvastatin treatment was associated with effective prevention of postoperative atrial fibrillation and cardiac mortality in patients aged over seventy years after CABG, as well as two different doses and duration of atorvastatin treatment in this surgical cohort. PATIENTS AND METHODS Data collection and patient selection We retrospectively evaluated consecutive 1890 patients (above the age of 70) attended to the Ankara Ataturk Education and Research Hospital, Cardiovascular Surgery Department from June 2004 to April 2012 and who underwent CABG. A total of 425 eligible patients were selected for the study. All patients who would have the coronary bypass surgery and no AF before surgery were included in this study. Patients with documented preoperative AF and associated cardiac surgery were excluded from the study. Routine electrocardiograms were obtained before the operation, on admission to the intensive care unit, for the first 72 hours after the surgery and every day thereafter until hospital discharge. Patients were divided into two groups: Atorvastatin Group (n=124, 29.17%) and No-statin Group (n=301, 70.82%). Patients in the Atorvastatin group received two different doses (20 and 40 mg) of atorvastatin starting within several weeks before the operation and throughout the postoperative period. The two groups were matched by propensity score analysis in terms of atrial fibrillation development and cardiac mortality. Primary and secondary end points The primary end points were the development of postoperative AF and cardiac mortality during the hospital stay. Postoperative AF was indicated as lasting for more than 10 minutes with symptoms. Cardiac mortality was defined as inhospital-mortality. Secondary end points were dose and duration of atorvastatin. Dose table for usage of atorvastatin was classified as four different categories: 10 mg, 20 mg, 40 mg and 80 mg. Time table for atorvastatin was analyzed according to the duration of the statin usage: 1 week, 1-2 weeks and > 2 week. In our institution, we prefer to use amiodarone in the management of postoperative AF after coronary surgery, electrical cardioversion being reserved only in those patients unresponsive to amiodarone treatment or having hemodynamic compromise. Considering embolic events, our routine practice was to start anticoagulation treatment with enoxaparin in all patients postoperatively. Operative procedure All patients underwent isolated CABG surgery and performed using conventional procedures. Induction and maintenance of anesthesia were similar for all patients and consisted of fentanyl, midazolam and pancuronium bromide. All operations were done with a median sternotomy incision. Cardiopulmonary bypass was performed in a standard fashion with the use of a hollow fiber membrane oxygenator (Dideco; Sorin Group, Mirandola, Italy) and a roller pump (Stöckert; Sorin Group, Mirandola, Italy), with high ascending aortic cannulation added to right atrium cannulation. Cardioplegic arrest was achieved with cold blood cardioplegia infused into the ascending aorta. Moderate temperature was about 32 ºC. Distal anastomosis was performed under the cross-clamp and proximal anastomosis was completed under the side-clamp. Intra-aortic ballon counterpulsation was inserted into the patients who had hemodynamic instability in the perioperative period. 191

90 Medicinski Glasnik, Volume 12, Number 2, August 2015 Statistical analysis Table 1. Demographic variables for atorvastatin and no statin groups Variables* Variables* AF (n=48) no-af (377) p Age 74.67± ± Females 30 (62.5%) 245 (65%) Diabetes Mellitus 11 (22.9%) 141 (37.4%) Hypertension 32 (66.7%) 222 (58.9%) Chronic obstructive pulmonary disease 13 (27.1%) 86 (22.8%) Creatinine 1.29± ± Other medications ß-blockers 35 (72.9%) 279 (74%) Angiotension converting enzyme inhibitors 18 (37.5%) 141(37.4%) Calcium channel blockers 9 (18.8%) 49 (13%) Cardiac status Class III-IV angina 21 (43.7%) 125 (33.2%) New York Heart Association class 10 (20.8%) III-IV 75 (19.9%) Previous myocardial infarction 7 (14.6%) 57 (15.1%) Ejection fraction < (25%) 72 (19.1%) Body surface area 1.73± ± Emergency 1 (2.1%) 4 (1.1%) Cross-clamp time (minutes) (11.14%) (13.99%) Cardiopulmonary bypass time (minutes) 80.23± ± Inotropic support 38 (79.2%) 298 (79.1%) Intra-aortic balloon counterpulsation 8 (16.7%) 41 (10.9%) Exitus 1 (2.1%) 11(2.9%) Atorvastatin-Group (n=124) No-statin- Group (n=301) Age 74.4± ± Females 86 (69.4%) 189 (62.8%) Diabetes Mellitus 42 (33.9%) 110 (36.5%) Hypertension 80 (64.5%) 174 (57.8%) Chronic obstructive pulmonary disease 30 (24.2%) 69 (22.9%) Creatinine 1.21± ± Other medications ß-blockers 104 (83.9%) 210 (69.8%) Angiotension converting enzyme inhibitors 66 (53.2%) 93 (30.9%) <0.001 Calcium channel blockers 26 (21%) 32 (10.6%) Cardiac status Class III-IV angina 69 (55.6%) 77 (25.6%) <0.001 New York Heart Association class III-IV 21 (16.9%) 64 (21.2%) Previous myocardial infarction 13 (10.5%) 51 (16.9%) Ejection fraction < (27.4%) 50 (16.6%) Body surface area 1.76± ± Emergency 3 (2.4%) 2 (0.7%) Cross-clamp time (minutes) 48.23± ± Cardiopulmonary bypass time (minutes) 82.1± ± Inotropic support 80 (64.5%) 256 (85%) <0.001 Intra-aortic balloon counterpulsation 25 (20.2%) 24 (8%) <0.001 Exitus 11 (8.9%) 11 (3.7%) *Categorical data are numbers (percentage); continuous data are means ± standard deviation. p Variables with a significance level of less than 0.2 were entered into a multivariable logit regression model and predictors of atorvastatin-group membership were identified. The propensity score matching approach is an alternative approach to address the potential selection bias (endogeneity) in the treatment effects. Predictors for inclusion in the atorvastatin-group, as identified by multivariate regression analysis of co-variables, followed by logistic regression, were used to create the propensity score model to adjust outcomes. Univariate analysis of all patients was also done for the development (or not) of postoperative atrial fibrilation (PAF). Variables that were found to have a value of p<0.20 in the univariate analysis were examined by multivariate logistic regression to determine predictors of PAF. Logistic regression with forward elimination determined the most important denominators for the development of PAF or not. Student t-test requires that the continuous variable should be normally distributed, but Mann- Whitney U test does not assume that the continuous variable is normally distributed. It only assumes that the variable is at least ordinal. Continuous and/or ordinal variables in our data do not satisfy the normality. Then, Mann-Whitney U test was used for continuous and/or ordinal variables to compare two groups. Chi-square test assumes the expected value of each cell is five or higher, but the Fisher s exact test has no such assumption and can be used regardless of how small the expected frequency is. Then, chi-square test was used for categorical variables if the expected value of each cell is five or higher in the data. In addition, Fisher s exact test was conducted in the case of violations of this assumption for some categorical variables in our data. Values of p<0.05 were considered statistically significant. RESULTS Patients characteristics The mean patient age was 74.4±3.2 years in Atorvastatin-Group versus 73.99±3.78 years in Nostatin-Group at the time of surgery (p=0.056). Females surprisingly were higher in both groups; 86 (69.4%) patients in Atorvastatin-Group versus 189 Table 2. Demographic variables for all patients with and without atrial fibrillation (AF) *Categorical data are numbers (percentage); continuous data are means ± standard deviation 192

91 Kunt et al. Preoperative statin treatment Table 3. Propensity score models for atorvastatin and atrial fibrillation: multivariate logistic regression, stepwise forward elimination Variables* Std. error Logit coefficient Exponential 95 % CI p coefficient For atorvastatin Age Females Hypertension Previous cerebrovascular accident Previous Urea ß-blockers Angiotension converting enzyme inhibitors <0.001 Calcium channel blockers Class III-IV angina <0.001 Previous myocardial infarction Ejection fraction < Three vessel disease Bilateral carotid stenosis Constant ACE inhibitors <0.001 ß-blockers Calcium channel blockers Class III-IV angina <0.001 Three vessel disease <0.001 Bilateral carotid stenosis Constant <0.001 For atrial fibrillation Three vessel disease Five-vessel bypass Constant <0.001 * Robust standard errors. (62.8%) patients in No-statin-Group (p =0.198) Atorvastatin-40 mg was used in over half of the patients, used in 68 (54.83%) patients who took atorvastatin. Of the patients, postoperative AF occurred in 9 (13.2%) patients (Adjusted OR=0.82; confidence interval= ; p=0.669). Nearly half of the patients (n=58, 46.77%) used atorvastatin between 1-2 weeks before surgery. Of the patients, postoperative AF occurred in 6 (10.3%) patients in this period (Adjusted OR=0.53; confidence interval= ; p=0.238) (Table 2). Postoperative atrial fibrillation Patient characteristics, procedural variables, and postoperative characteristics for patients with and without were similar. Postoperative AF occurred in 48 (11.29%) patients in all patients. The mean patient age for AF was 74.67±3.77 years in Atorvastatin-Group versus 74.04±3.60 years in No-statin-Group at the time of surgery (p=0.209) (Table 3). Propensity score analysis for atorvastatin and for AF has shown that three-vessel disease (Logit coefficient: , Exponential coefficient: 0.212, 95 % confidence interval: , p=0.035) and five-vessel bypass (Logit coefficient: 0.856, Exponential coefficient: 2.354, 95 % confidence interval: , p=0.050) were found as an independent predictor for the development of AF (Table 4). Table 4. Dose and time of atorvastatin for Atorvastatin group Atorvastatin- Group (n=124) AF Adjusted OR* CI* p* Dose (18.54%) 3 (10.3%) (25%) 4 (12.9%) (54.83%) 9 (13.2%) (1.61%) 1(50%) Time 1 week 42 (33.87%) 6(14.3%) weeks 58 (46.77%) 6 (10.3%) > 2 weeks 24 (19.35%) 5 (20.8%) Şub *adjusted for propensity score; AF, atrial fibrillation; CI, confidence interval Cardiac mortality Cardiac death occurred in 22 (5.2%) patients. Although mortality was statistically significant in the no-statin-group comparing to statin group, the reasons of mortality were independent from the statin therapy. The reasons of the mortality were low cardiac output (1.4%), unable to wean cardiopulmonary bypass (0.7%), respiratory failure (0.7%), major cerebrovascular event (0.47%), gastrointestinal bleeding (0.47%), intestinal ischemia (0.47%), failure of left internal thoracic artery (0.47%), ventricular fibrillation (0.23%), and bleeding from the mediastinal space (0.23%). DISCUSSION Meta-analyses have demonstrated that some pharmacological agents and biatrial pacing reduced postoperative AF (10,11). Only amiodarone and beta-blockers have shown to be effective for the management of postoperative AF as advised recently by the American College of Cardiology/ American Heart Association/European Society of Cardiology guidelines. Subsequently, some studies reported that there is a relationship between preoperative statin use and reduced postoperative AF (12-14). 193

92 Medicinski Glasnik, Volume 12, Number 2, August 2015 Relationship between preoperative statin use and rates of reduced postoperative was first reported by ARMYDA-3 trial (7). Two hundred patients who underwent coronary bypass were randomized to either atorvastatin (40 mg/d, n=101) or placebo (n=99) starting 7 days preoperatively. There was a 61% reduction in postoperative AF in patients who received statins; AF occurred in 35% of patients with statins and 57% of patients with placebo (p=.017). However, some authors have suggested that a dose response relationship with statins and postoperative AF may also exist (15-17). Lertsburapa et al (15) conducted a nested case control study with data from the randomized, controlled Atrial Fibrillation Suppression Trials I, II, and III and found that higher statin doses (atorvastatin 40 mg/d) were associated with greater reduction in postoperative AF than were lower doses. This finding was later corroborated by observational studies conducted by Kourliouros (16) and Mathani (17) et al. One of the findings of our study was that preoperative statin treatment was not associated with the reduction of postoperative AF in the patients. The other one was that there was no relationship between dose and duration of statin therapy for the development of postoperative AF in this specific surgical cohort. As it is known, advanced age is a major risk factor for the development postoperative AF after cardiac surgery (3,18,19). Levy and colleagues (20) suggested that age is a very powerful predictor of postoperative AF. Actually, there is limited data whether statin treatment has beneficial effects in advanced age on postoperative AF after the coronary surgery. The Multicenter Study of Perioperative Ischemia Research Group and investigators of the Ischemia Research and Education Foundation have published the prospective study performed in 70 hospitals on 4 continents (1) including more than 5,000 patients undergoing CABG operations with or without valve surgery on cardiopulmonary bypass: patients with postoperative AF were significantly older (67.8 years versus 61.8 years), and a significantly larger number had a history of AF (14.6% versus 6.0%). The incidence of postoperative AF was 11.29% in the present study population after coronary artery bypass grafting. Postoperative AF is thought to be mostly benign (21,22), it increases late mortality after isolated coronary surgery only (23). Kalavrouziotis and coworkers (24) concluded the same in a large study on postoperative AF in cardiac surgery patients after multivariate analysis and propensity score matching. When considering early cardiac mortality, it is really hard to combine the effects of these two clinical positions on cardiac death: preoperative statin therapy and postoperative AF. In the study of Villareal and colleagues (25), postoperative AF was a significant predictor of early death after adjusting risk factors. A large number of studies (3,8,9,22,24) show significantly higher incidence of early death in patients with postoperative AF after coronary bypass surgery or cardiac operations, but none of these studies identified postoperative AF as an independent predictor of early mortality. In the present study we have reported in-hospital cardiac mortality of 8.9% in the Atorvastatin-Group versus 3.7% in the No-statin-Group (p=0.027); cardiac death in patients with and without AF occurred at approximately 2.1% in the Atorvastatin-Group versus 2.9% in the No-statin-Group. Despite a small number of patients in the present study, we cannot rule out widespread use of statins in patients undergoing coronary artery bypass grafting to prevent postoperative atrial fibrillation. We cannot exclude the possibility that ß-blockers and ACE inhibitors attenuated the benefits in our study or that the results were due to chance or population differences. Two groups were not homogenous. No-statin-Group had higher patient population compared to Atorvastatin-Group. Over a half of the patients were females in contrast to standard population of coronary surgery. Postoperative atrial fibrillation is a frequent complication of cardiac operations and may result in serious cardiac adverse events including cardiac mortality. Although further work is necessary before any definitive recommendation, omitting statin drugs is not found to be associated with reduced postoperative atrial fibrillation and cardiac mortality in patients undergoing isolated coronary artery bypass grafting above the age of seventy years in the perioperative period. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. 194

93 Kunt et al. Preoperative statin treatment REFERENCES 1. Mathew JP, Fontes ML, Tudor IC, Ramsay J, Duke P, Mazer CD, et al. Investigators of the Ischemia Research and Education Foundation; Multicenter Study of Perioperative Ischemia Research Group. A multicenter risk index for atrial fibrillation after cardiac surgery. JAMA 2004; 291: Mariscalco G, Klersy C, Zanobini M, Banach M, Ferrarese S, Borsani P. Atrial fibrillation following coronary artery bypass graft surgery: predictors, outcomes, and resource utilization. Multi Center Study of Perioperative Ischemia Research Group. JAMA 1996; 276: Aranki SF, Shaw DP, Adams DH, Rizzo RJ, Couper GS, VanderVliet M. Predictors of atrial fibrillation after coronary artery surgery. Current trends and impact on hospital resources. Circulation 1996; 94: Hashimoto K, Ilstrup DM, Schaff HV. Influence of clinical and hemodynamic variables on risk of supraventricular tachycardia after coronary artery bypass. J Thorac Cardiovasc Surg 1991; 101: Butler J, Harriss DR, Sinclair M, Westaby S. Amiodarone prophylaxis for tachcardias after coronary artery surgery: a randomized, doubleblind, placebo controlled trial. Br Heart J 1993; 70: Wendy T. Chen, Guru M. Krishnan, Nitesh Sood, Jeffrey Kluger, Craig I Coleman. Effect of statins on atrial fibrillation after cardiac surgery: A duration- and dose-response meta-analysis. J Thorac Cardiovasc Surg 2010; 140: Patti G, Chello M, Candura D, Pasceri V, D Ambrosio A, Covino E, Randomized trial of atorvastatin for reduction of postoperative atrial fibrillation in patients undergoing cardiac surgery: results of the ARMY- DA-3 (Atorvastatin for Reduction of MYocardial Dysrhythmia After cardiac surgery) study. Circulation 2006; 114: Mariscalco G, Klersy C, Zanobini M, Banach M, Ferrarese S, Borsani P. Atrial fibrillation after isolated coronary surgery affects late survival. Circulation 2008; 118: Almassi GH, Schowalter T, Nicolosi AC, Aggarwal A, Moritz TE, Henderson WG. Atrial fibrillation after cardiac surgery: a major morbid event? Ann Surg 1997; 226: Burgess DC, Kilborn MJ, Keech AC. Interventions for prevention of post-operative atrial fibrillation and its complications after cardiac surgery: a metaanalysis. Eur Heart J 2006; 27: Crystal E, Connolly SJ, Sleik K, Ginger TJ. Yusuf S. Interventions on prevention of postoperative atrial fibrillation in patients undergoing heart surgery: a meta-analysis. Circulation 2002; 106: Dotani MI, Elnicki DM, Jain AC, Gibson CM. Effect of preoperative statin therapy and cardiac outcomes after coronary artery bypass grafting. Am J Cardiol 2000; 86: Marín F, Pascual DA, Roldán V, Arribas JM, Ahumada M, Tornel PL. Statins and postoperative risk of atrial fibrillation following coronary artery bypass grafting. Am J Cardiol 2006; 97: Ozaydin M, Dogan A, Varol E, Kapan S, Tuzun N, Peker O. Statin use before by-pass surgery decreases the incidence and shortens the duration of postoperative atrial fibrillation. Cardiology 2006; 107: Lertsburapa K, White CM, Kluger J, Faheem O, Hammond J, Coleman CI. Preoperative statins for the prevention of atrial fibrillation after cardiothoracic surgery. J Thorac Cardiovasc Surg 2008; 135: Kourliouros A, De Souza A, Roberts N, Marciniak A, Tsiouris A, Valencia O. Dose-related effect of statins on atrial fibrillation after cardiac surgery. Ann Thorac Surg 2008; 85: Mithani S, Akbar MS, Johnson DJ, Kuskowski M, Apple KK, Bonawitz-Conlin J. Dose dependent effect of statins on postoperative atrial fibrillation after cardiac surgery among patients treated with beta blockers. J Cardiothorac Surg 2009; 4: Creswell LL, Schuessler RB, Rosenbloom M, Cox JL. Hazards of postoperative atrial arrhythmias. Ann Thorac Surg 1993; 56: Mathew JP, Parks R, Savino JS, Friedman AS, Koch C, Mangano DT. Atrial fibrillation following coronary artery bypass graft surgery. JAMA 1996; 276: Levy D, Kannel WB. Postoperative atrial fibrillation and mortality: do the risks merit changes in clinical practice? J Am Coll Cardiol 2004; 43: Maisel WH, Rawn JD, Stevenson WG. Atrial fibrillation after cardiac surgery. Ann Intern Med 2001; 135: Ahlsson A, Bodin L, Fengsrud E, Englund A. Patients with postoperative atrial fibrillation have a doubled cardiovascular mortality. Scand Cardiovasc J 2009; 12: Giovanni Mariscalco and Karl Gunnar Engström. Postoperative atrial fibrillation is associated with late mortality after coronary surgery, but not after valvular surgery. Ann Thorac Surg 2009; 88: Kalavrouziotis D, Buth KJ, Ali IS. The impact of new-onset atrial fibrillation on in-hospital mortality following cardiac surgery. Chest 2007; 131: Villareal RP, Hariharan R, Liu BC, Kar B, Lee VV, Elayda M. Postoperative atrial fibrillation and mortality after coronary artery bypass surgery. J Am Coll Cardiol 2004; 43:

94 ORIGINAL ARTICLE Efficacy of chronic statin therapy on major cardiac events after coronary artery bypass grafting: low-dose versus high-dose Ayşegül Kunt 1, Sedat Özcan 2, Aslihan Küçüker 3, Dolunay Odabaşi 1, Alper Sami Kunt 1 1 Department of Cardiovascular Surgery, School of Medicine, 100.Yil University, Van, 2 Department of Cardiovascular Surgery, School of Medicine, 18 Mart University, Çanakkale, 3 Department of Cardiac Surgery, Ataturk Education and Research Hospital, Ankara; Turkey ABSTRACT Aim To investigate whether chronic statin treatment after coronary artery bypass grafting (CABG) protects patients from major cardiac events and provides percutaneous coronary intervention (PCI) free survival. Corresponding author: Sedat Özcan Department of Cardiovascular Surgery, School of Medicine, Canakkale On Sekiz Mart University Kepez, Çanakkale, Turkey Phone: ; Fax: ; sedatozcan78@hotmail.com Original submission: 21 January 2015; Revised submission: 13 March 2015; Accepted: 18 March doi: / Methods A total of 232 patients with previous CABG and chronic statin therapy were selected retrospectively and were divided into two groups according to a dosage of atorvastatin per day, e. g., 20 mg or 40 mg. Groups were compared for the major cardiac events and freedom from PCI by Kaplan Meier analysis as the primary end point. Patency of grafts including left internal thoracic artery (LITA) and saphenous vein (SVG) and progression of non-grafted native vessel disease were also evaluated as secondary end points. Results Cardiac mortality, periprocedural myocardial infarction (MI), target vessel revascularization and percutaneous coronary intervention free survival were as follows: 2.9% versus 2.1% (p=1.000); 16.1% versus 21.1% (p=0.331); 56.93% versus 52.63% (p>0.005); 58.4% versus 63.2% (log-rank test; p= 0.347) in atorvastatin 20 mg and atorvastatin 40 mg groups, respectively. However, these results were not statistically significant between two groups (p>0.005). Patency of openness of grafts including LITA and SVG and progression of non-grafted native vessel disease were similar between groups (p=0.112, p=0.779, p=0.379 and p=0.663, respectively). Conclusion Low-dose long-term statin treatment had similar outcomes on major cardiac events and identical rate of freedom from percutaneous coronary intervention after coronary artery bypass grafting compared with high-dose long-term statin treatment. It is better to start from low dose statin treatment after surgical interventions. Key words: chronic statin treatment, major cardiac events, coronary bypass grafting Med Glas (Zenica) 2015; 12(2):

95 Kunt et al. Major cardiac events after coronary bypass INTRODUCTION Coronary artery bypass grafting (CABG) is a therapeutic choice for advanced coronary artery disease. However, patients are still at significant risk for postoperative major cardiac events (1). Although statins protect patients from subsequent coronary ischemic events, optimal selection of drug and ideal dosage are still open to debate (2-3). Moreover, it is not clear whether longterm statin therapy would have improved clinical outcomes in patients after CABG (3). The aim of this study is to show low-dose versus high dose of non-stop atorvastatin therapy after CABG would keep the patients away from major cardiac events and provide PCI-free survival. Cardiac death, periprocedural myocardial infarctus (MI), target vessel revascularization and percutaneous coronary intervention (PCI), free survival were considered as primary end points. Patency of grafts including left internal thoracic artery (LITA) and saphenous vein graft (SVG) and progression of coronary ischemic disease in native coronary arteries after CABG were also evaluated. PATIENTS AND METHODS Patient selection This study was performed retrospectively. A total number of 13,558 patients who underwent conventional coronary angiography at Ankara Ataturk Education and Research Hospital between 2009 and 2012 (inclusion criteria were stable angina and indication to coronary angiography, ST and non ST-segment elevation acute MI; exclusion criteria were bypass of left anterior descending artery other than LITA, free LITA) were initially reviewed for patients with previous CABG. Patients were then reviewed for postoperative PCI. A total number of 289 patients fulfilling the inclusion criteria were re-evaluated; 57 patients (19.7%) met the exclusion criteria: not on statin therapy (n=35, 12.1%), for free LITA (n=13), bypass of left anterior descending artery other than LITA (n=9). Finally, 232 patients with previous CABG and postoperative PCI and chronic statin therapy represented the study population. These eligible patients were divided into two groups according to the statin dose: as 20 mg or 40 mg atorvastatin per day Percutaneous coronary intervention Percutaneous coronary interventions were performed with the standard technique; patients were pre-treated with aspirin (100 mg/day) and clopidogrel (600-mg loading dose at least 3 h before the procedure) (1-3). Following PCI, aspirin (100 mg/day) was continued indefinitely, whereas clopidogrel (75 mg/day) was administered for at least 1 month (12 months in patients treated for acute coronary syndrome or receiving stents). Primary end points Cardiac death was defined as in-hospital 30-day mortality. Periprocedural MI was evaluated as elevated cardiac biomarkers (troponin or CK- MB) and electrocardiographic findings. Target vessel revascularization was defined as PCI, which was divided into 3 separate periods and target vessels. Target vessel non-revascularization was described as PCI-free survival. Secondary end points Stenosis of over 80% of LITA and over 60% of SVG were evaluated as occluded. Progression of the coronary ischemic disease in native coronary arteries was classified according to the degree of the stenosis. Stenosis was classified as over and above 50%. These native coronary arteries had not been bypassed earlier. Statistical analysis Univariate analysis was performed to examine differences in variables between patients receiving atorvastatin 20 and 40 mg groups. Categorical variables between groups were evaluated by using the chi-square test or Fisher s exact test. Continuous variables were conducted by using Mann-Whitney U test. Value of p <0.05 was considered statistically significant. Moreover, PCIfree survival analysis was performed by Kaplan- Meier analysis and compared by the log rank test in both groups. Stent insertion was considered as the failure event (hazard) and no-stent insertion (PCI-free) was considered as the survival. RESULTS Patients characteristics Both groups were similar at the time of PCI, except for recent ex-smoker and using clopido- 197

96 Medicinski Glasnik, Volume 12, Number 2, August 2015 grel. The group of atorvastatin-40 mg had higher recent ex-smoker (patients who used to smoke but quitted recently) and taking clopidogrel (p<0.001 and p=0.004 respectively) (Table 1). Table 1. Demographic characteristics and other medical therapy of atorvastatin-20 mg and atorvastatin-40 mg groups No (%) of patients Variables Atorvastatin-20 mg group (n=137) Atorvastatin-40 mg group (n=95) Age ± ± Females 33 (24.1%) 20 (21.1 %) Diabetes mellitus 54 (39.4%) 30 (31.6 %) Hypertension 72 (52.6%) 44 (46.3 %) Chronic obstructive pulmonary disease 10 (7.3%) 4 (4.2 %) Peripheral arterial disease 6 (4.4%) 6 (6.3 %) Dialysis 3 (2.2%) 1 (1.1%) Total cholesterol ± ± Triglyceride ± ± High density lipoprotein ± ± Low density lipoprotein ± ± Cigarette smoker 19 (13.9%) 12 (12.6%) Recent ex-smoker 10 (7.3%) 23 (24.2%) <0.001 Ex-smoker 108 (78.8%) 60 (63.2%) ß-blocker 119 (86.9%) 83 (87.4%) Angiotension converting enzyme 97 (70.8%) 77 (81.1%) Ca+2 channel blocker 43 (31.4%) 30 (31.6%) Nitrate 132 (96.4 %) 91 (95.8%) Clopidogrel 68 (49.6%) 65 (68.4%) Cardiovascular profiles of both groups were also similar, related with the Canadian Cardiovascular Society classification and unstable angina pectoris (USAP). The group of Atorvastatin-40 mg had higher Canadian Cardiovascular Society classification and USAP (p=0.005 and p<0.001 respectively) (Table 2). p Operative characteristics, follow-up, patency of grafts and stenosis of non-grafted native coronary arteries and PCI of target vessel(s) were similar for both groups (Tables 3-5 ) During the follow-up, conventional coronary angiography was performed in all patients at the first hospitalization, in 43 patients at the second hospitalization and in five patients at the third hospitalization (Table 3). Table 3. Operative characteristics of atorvastatin-20 mg and atorvastatin-40 mg groups Atorvastatin-20 Atorvasta- Variables* Group tin-40 Group p (n=137) (n=95) Number of distal anastomosis 2.32 ± ± Sequential bypass 18 (13.1%) 15 (15.8%) Number of venous graft 1.24 ± ± Number of arterial graft 1.08 ± ± Distal insertion site other than left anterior descending artery (No (%) of patients) Diagonal artery 30 (21.9%) 20 (21.1%) Optional diagonal artery 1 (0.7%) 3 (3.2%) Circumflex artery 82 (59.9%) 61 (64.2%) Right coronary artery 69 (50.4%) 45 (47.4%) RCAPDA 13 (9.5%) 7 (7.4%) RCAPL 1 (0.7%) 1 (1.1%) *Categorical data are numbers (percentage), continuous data are means ± standard deviation. RCAPD, right coronary artery posterior descending artery; RCAPL, right coronary artery posterolateral artery Cardiac death occurred in four (2.9%) patients in the group of atorvastatin-20 mg and in two (2.1%) patients in the group of atorvastatin-40 mg (p=1.000) (Table 4). Periprocedural MI happened in 22 (16.1%) patients in the group of atorvastatin-20 mg and in 20 (21.1%) patients in the group of atorvastatin-40 mg (p=0.331) (Table 4). Table 2. Cardiovascular profile of atorvastatin-20 mg and atorvastatin-40 mg groups No (%) of patients Atorvastatin-20 Atorvasta- Variables* Group tin-40 Gro- p (n=137) up (n=95) CCS III/IV 86 (62.8 %) 76 (80%) NYHA III/IV 26 (19%) 13 (13.7%) Chronic heart failure 13 (9.5%) 4 (4.2%) Cardiogenic shock 5 (3.6%) Cardiopulmonary resuscitation 5 (3.6%) Unstable angina pectoris 44 (32.1 %) 53 (55.8 %) <0.001 Stable angina pectoris 64 (46.7 %) 27 (28.4%) Previous myocardial infarction 18 (13.1%) 22 (23.2%) Previous PCI 7 (5.1%) 4 (4.2%) Ejection fraction < (45.3%) 43 (45.3%) Left main coronary artery 18 (13.1%) 13 (13.7%) *Categorical data are numbers (percentage); continuous data are means ± standard deviation. CCS, Canadian Cardiovascular Society; NYHA, New York Heart Association; PCI, percutanous coronary intervention. Table 4. Follow-up of atorvastatin-20 mg and atorvastatin-40 mg groups Variables* Atorvastatin-20 Atorvastatin-40 Group (n=137) Group (n=95) p Number of outpatient control after CABG ± ± Number of hospitalization 1.88 ± ± Time interval 6.49 ± ± Periprocedural myocardial infarction 22 (16.1%) 20 (21.1%) Mortality 4 (2.9%) 2 (2.1%) *Categorical data are numbers (percentage), continuous data are means ± standard deviation. CABG, coronary artery bypass grafting Time interval between CABG and last coronary angiography was 6.49 ± 4.47 years and 6.33 ± 4.58 years in the group of atorvastatin-20 mg and 40 mg, respectively. The total number of PCI was 56.93% in 78 patients in the group of atorvasta- 198

97 Kunt et al. Major cardiac events after coronary bypass tin-20 mg and 52.63% in 50 patients in the group of atorvastatin-40 mg (p>0.005) Primary end points The total number of intervention was 61 (44.52%) versus 36 (37.89%) during the first procedure, 12 (8.75%) versus 10 (10.52%) during the second procedure and 5 (3.64%) versus 4 (4.21%) during the third procedure in the group of atorvastatin 20 and in the group of atorvastatin 40 mg respectively. PCI-free survival was 58.4% in the group of atorvastatin-20 mg and 63.2% in the group of atorvastatin-40 mg in this period (p=0.347) Secondary end points Patent and occluded LITA were observed in 200 (86.2%) and in 32 (13.79%) out of 232 patients in both groups (atorvastatin-20 mg and atorvastatin-40 mg), respectively (p=0.112). Patent SVG was visualized in 186 (80.17%) patients of both groups (p=0.779). Stenosis of circumflex artery over 50% obstruction was seen in 38 (33.5%) patients (p=0.379) in both groups. Stenosis of right coronary artery over 50% was seen in 60 (52.2%) patients (Table 5). Table 5. Patency of grafts and stenosis of native coronary arteries of atorvastatin-20 mg and atorvastatin-40 mg groups Variables* Atorvastatin-20 Group (n=137) Atorvastatin-40 Group (n=95) LITA-patent 114 (83.2%) 86 (90.5%) LITA-occluded 23 (16.8%) 9 (9.5%) SVG-patent 109 (79.6%) 77 (81.1%) Cx >50% stenosis 20 (14.6%) 18 (18.9 %) RCA >50% stenosis 34 (24.8%) 26 (27.4 %) *Categorical data are numbers (percentage), continuous data are means ± standard deviation. Cx, circumflex artery; LITA, left internal thoracic artery; RCA, right coronary artery; SVG, saphenous vein graft DISCUSSION Coronary artery bypass grafting is an effective treatment for patients with coronary artery disease (4,5). However, hyperlipidemia can cause recurrent ischemic cardiac events in this patient population in the postoperative period (6). Guidelines by the American College of Cardiology and American Heart Association recommended statins for CABG patients with LDL concentrations greater than 100 mg/dl (7). Although statins reduce major cardiac events after CABG, there is no available data for PCI-free survival in patients with long-term statin intake in the postoperative period. We also sought primarily the influence of p different doses of atorvastatin on major cardiac events and secondary patency of LITA and SVG and progression of native vessel disease in longterm period after CABG. The Post Coronary Artery Bypass Graft Trial was designed to compare the effects of 2 lipid-lowering regimens in patients who had CABG (8-10). The primary endpoint had been planned as cardiac death or nonfatal acute MI and found 15.1% in the aggressive strategy group and 20.3% in the moderate strategy group. The investigators could not have shown significant difference between two strategies in the occurrence of death. We could not show any difference between groups in the occurrence of cardiac mortality in patients after CABG either. The CARE (In the Cholesterol and Recurrent Events) trial included 1,091 patients (pravastatin (n=527) and placebo (n=564)) who had previously undergone CABG for a mean period of 5 years (11). In our study, follow-up period was: 6.49 ± 4.47 and 6.33 ± 4.58 in the Group of Atorvastatin 20 and 40 mg respectively. The authors demonstrated that pravastatin produced a statistically significant reduction (24%) in the relative risk of a composite endpoint of fatal coronary event or nonfatal acute MI relative risk of a composite endpoint of fatal coronary heart disease death (33%) or nonfatal acute MI (absolute risk 9.1% versus 12.9% in the placebo group), by 41% of the incidence of coronary death (4.6% versus 7.8%), and by 35% total mortality (8% versus 12.4%). In the trial, the authors divided MI as fatal and nonfatal; we reported periprocedural MI together in our study. The rates of total and cardiac mortality had been given separately in the trial. We excluded the death from non-cardiac reasons in order not to affect the cardiac results. Some clinical data indicated that pretreatment with statins may significantly reduce periprocedural complications and major adverse cardiac events in patients undergoing PCI (13-15). Patti et al. performed a collaborative meta-analysis using data from 13 randomized studies in which patients received either high-dose statin (n=1.692) or no statin/low-dose statin (n=1.649) before PCI, with all patients receiving statin therapy after intervention (16). Our patient population had prior CABG, which means that patients received statin before PCI. The authors found that periprocedural MI was 199

98 Medicinski Glasnik, Volume 12, Number 2, August 2015 lower in the high-dose statin versus control group, which corresponds to a 44% risk reduction in the active-treatment arm. They also demonstrated that major adverse cardiac events within 30 days was significantly lower in the high-dose statin group, and 1-month major adverse cardiac events, excluding periprocedural events, were also reduced: the results were much more reliable as expected. In our study, we evaluated not only PCI but also PCIfree survival after surgery; both groups were similar for PCI and PCI-free survival under the statin treatment after CABG. We thought that PCI-free survival was higher in Atorvastatin-40 mg, because this group of patient had a higher incidence of unstable angina pectoris (USAP) Carrier et al analyzed the benefit of statin treatment on single and bilateral ITA grafts on longterm survival after CABG (17). The paper included patients. The authors reporteded that the patients with bilateral ITA grafts had an average 10-year-survival rate of 83% compared with 67% in patients with single ITA grafts. The authors also demonstrated that statin treatment caused a significant decrease in the long-term risk of death among patients who underwent single ITA grafting, but not in those with bilateral ITA grafting. However, they demonstrated that survival of statin-treated patients with single ITA grafts was similar to bilateral ITA patients. In our study, we only evaluated patency of LITA grafting in two groups separately, showing that patency of ITA grafting is appropriate with the literature in each group because over 90% of LITAs remain patent within 10 years after surgery. The CASCADE (Clopidogrel After Surgery for Coronary Artery Disease) trial was designed to evaluate the addition of clopidogrel (75 mg) to aspirin (162 mg) on the development of saphenous vein graft disease after CABG. The patients received statin therapy after operation achieved REFERENCES 1. Pasceri V, Patti G, Nusca A, Pristipino C, Richichi G, Di Sciascio G. ARMYDA Investigators. Randomized trial of atorvastatin for reduction of myocardial damage during coronary intervention: results from the ARMYDA (Atorvastatin for Reduction of MYocardial Damage during Angioplasty) study. Circulation 2004; 110: Patti G, Pasceri V, Colonna G, Miglionico M, Fischetti D, Sardella G, Atorvastatin pretreatment improves outcomes in patients with acute coronary an LDL level less than 100 mg/dl (18-20) and underwent angiography of the bypass grafts and the native coronary arteries; statin therapy was independently associated with improved graft patency. In our study, patients reached LDL level less than 100 mg/dl in both atorvastatin groups, but without improved graft patency of SVG between the groups. Our result of patency of SVG is in accordance with the literature in each group because within 10 years after surgery, only 60% of the SVGs remain patent and half of those that are patent have clinically important stenosis (5,21,22 ). CLAS (The Cholesterol Lowering Arteriosclerosis Study) showed that aggressive cholesterol reduction for a period of 2 to 4 years significantly reduces new lesion formation in native vessels (23). In our study, comparing old clinical reports of coronary angiograms with the last ones, and evaluating only the previously non-grafted vessels we could not find any difference between the atorvastatin groups for the stenosis over 50%. The major limitation of the present study is its retrospective and nonrandomized design. Furthermore, we are unable to obtain the third group of patients who had not received statins. Of course, there could be changes in the dosage of statin throughout the study. Essentially all patients should be prescribed long-term statin therapy independent from the dose to reduce cardiac events after CABG. This confirms earlier studies within a contemporary surgical population and supports the current clinical guidelines. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. syndromes undergoing early percutaneous coronary intervention: results of the ARMYDA-ACS randomized trial. J Am Coll Cardiol 2007; 49: Patti G, Colonna G, Pasceri V, Pepe LL, Montinaro A, Di Sciascio G. Randomized trial of high loading dose of clopidogrel for reduction of peri-procedural myocardial infarction in patients undergoing coronary intervention: results from the ARMYDA-2 (Antiplatelet therapy for Reduction of MYocardial Damage during Angioplasty) study. Circulation 2005; 111:

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100 ORIGINAL ARTICLE Incidence of endophthalmitis after intravitreal application of anti VEGF therapy at the University Clinical Center in Tuzla, Bosnia and Herzegovina Svjetlana Terzić, Adisa Pilavdžić, Amra Nadarević Vodenčarević Eye Clinic, University Clinical Centre Tuzla, Tuzla, Bosnia and Herzegovina ABSTRACT Aim To report the incidence of endophthalmitis following the use of intravitreal injection of anti- vascular endothelial growth factor (anti VEGF) therapy. Methods In this retrospective study a total of 986 intravitreal bevacizumab injections were applied between January 2008 and April 2015 at the University Clinical Center Tuzla, Bosnia and Herzegovina (B&H). Since January 2012, a total of 55 intravitreal ranibizumab injections were applied and since October 2014, 60 intravitreal aflibercept injections were applied to patients. Corresponding author: Amra Nadarević Vodenčarević Eye Clinic, University Clinical Center Tuzla Trnovac b.b., Tuzla, Bosnia and Herzegovina Phone: ; amra_nadarevic@hotmail.com Results Two cases of endophthalmitis following intravitreal injection of bevaciuzumab occurred and none after ranibizumab or aflibercept. The overall incidence of clinical endophtahlmitis was 0.2%. Conclusion The results suggest that a low rate of endophthalmitis can be achieved by means of a protocol. This is a very important study as it is the first of this kind in B&H that documents the incidence of endophthalmitis after intravitreal application. Currently, bevacizumab in B&H is most frequently used intravitreal anti-vascular endothelial growth factor due to very low price. Key words: bevacizumab, aflibercept, ranibizumab, complications of intravitreal application Original submission: 07 May 2015; Revised submission: 14 July 2015; Accepted: 18 July doi: / Med Glas (Zenica) 2015; 12(2):

101 Terzić et al. Endophthalmitis after VEGF intravitreal application INTRODUCTION Endophthalmitis is uncommon, but very severe ocular inflammatory process that can lead to blindness (1). During this process, inflammation affects vitreous cavity along with the retinal and uveal components of the eye. After eye surgery or intravitreal application of anti vascular endothelial growth factor (anti VEGF) post-operative endophtalmitis is possible to occur, and it can have two forms, either sterile or infectious (2). Since its beginning in 2004, intravitreal injections of anti VEGF in whole world have had a huge impact on treatment of variety of ocular conditions (3). The number of intravitreal injections is increasing in Bosnia and Herzegovina (B&H). Currently, at the University Clinic Center (UCC) in Tuzla, B&H, intravitreal injections have become a common route of administration of medications. At UCC Tuzla currently we are applying intravitreal injections bevacizumab, ranibizumab and aflibercept. This intravitreal injections are applied for a variety of conditions including complications of diabetic retinopathy, such as diabetic macular edema (DME) and as well other retinal-vascular disorders, such as branch retinal vein occlusion, central retinal vein occlusion and as well age related macular degeneration (AMD) (4-6). At this moment only ranibizumab and aflibercept are labeled for intravitreal use, while bevacizumab is currently also being used offlabel for the treatment of ocular disease. This off-label intravitreal injections of bevacizumab have been given for the treatment of neovascular and exudative ocular diseases since May 2005 (7). To our knowledge until today, from February 2004 bevacizumab has been approved by the US Food and Drug Administration (FDA) for treating patients with metastatic colorectal cancer (8). All over the world between 1997 and 2001, fewer than 5,000 intravitreal injections were performed annually, while more than 800,000 were performed in 2007 (9). To minimize any complication or infections after intravitreal application of anti VEGF several protocols have been proposed (10-11). Recent surveys have suggested that 40% of retina specialists use topical antibiotics before anti vascular endothelial growth factor intravitreal injections, and 86% use topical antibiotics after anti vascular endothelial growth factor intravitreal injections (12). The aim of this study was to investigate the incidence of endophthalmitis following the use of intravitreal injection of anti-vascular endothelial growth factor (anti VEGF) therapy. PATIENTS AND METHODS University Clinic Center of Tuzla is a single health institution equipped for the intravitreal application of anti VEGF therapy in north-eastern part of B&H. This is a single-center retrospective analysis of all intravitreal application at the UCC Tuzla. All patients who received intravitreal injections of bevacizumab, ranibizumab and aflibercept were included in this study. Patients receiving other intravitreal injections (including corticosteroids or antibiotics) were excluded. The indications for intravitreal injection included exudative age-related macular degeneration (AMD), choroidal neovascular membranes secondary to myopic degeneration, cystoid or diffuse macular edema from central and branch vein occlusions, and as well edema due to a diabetic complication. All intravitreal injections were recorded in doctor and nursing logbooks. In this study provisions of the Helsinki Declaration were followed. Before application all the patients who were treated with 0.05 ml injection containing 1.25 mg of bevacizumab, 0.3 mg /0.05 ml ranibizumab or 2.0 mg/0.05 ml aflibercept, underwent complete eye examination. The complete eye examination included determination of best-corrected visual acuity, slit-lamp examination, intraocular pressure measurement and retinal thickness measurement by optical coherence tomography. After written informed consents were obtained, all patients were treated. The informed consent was given to all patients due the risks of intravitreal injection which included pain, bleeding, retinal detachment, cataract, increased transient IOP, infections and sterile endophthalmitis. Injections were performed by retinal specialist in the operation room. The medications were given under aseptic conditions. Lids and conjunctiva were prepared with 5% povidone ioidine, followed by the placement of an eyelid speculum. Intravitreal injections were injected with a needle in infero-temporal quadrant through pars plana (3,5 4,0 mm from limbus) into the vitreous cavity. Patients were instructed to administer topical Maxitrol (Alcon) 3 times daily for 5 days. All applications were performed as an outpatient pro- 203

102 Medicinski Glasnik, Volume 12, Number 2, August 2015 cedure. Patients were told to return to the hospital immediately if visual disturbance, pain, or redness of the eyes were noticed. Generally all the patients following intravitreal injections were followed up in the clinic usually at 1-3 monthly intervals. Clinical diagnosis of endophthalmitis was made on the basis of presence of anterior chamber reaction, keratitic precipitates, hypopion, fibrin and/ or posterior synechia. Ultrasound examination was performed with (10MHz transducer, UltraScan, ALCON Inc, Fort Worth, TX, USA). RESULTS In a period of 7 years a total of 986 patients with intravitreal bevacizumab injection, 1.25 mg/0.05 ml was treated. Since 2012 fifty-five patients had been treated with intravitreal ranibizumab injection 0.3 mg /0.05 ml and since October 2014 sixty patients had been treated with intravitreal aflibercept injection 2.0 mg/0.05 (Table1). Table 1. Total number of intravitreal injections of anti VEGF at UCC Tuzla and the number of endophthalmitis cases that occurred after intravitreal injection Type of anti VEGF Total number of intravitreal injections Number of endophthalmitis cases after intravitreal injections RANIBIZUMAB 55 0 BEVACIZUMAB AFLIBERCEPT 60 0 The overall incidence of clinical endophtahlmitis was 0.2%. Injections were administered in an operating room. Patients presented with decreased vision, pain and red eye. Two cases of endophthalmitis following intravitreal injection of bevaciuzumab occurred and none after ranibizumab or aflibercept. Both patients with endophthalmitis were males. The mean interval between intravitreal injections and onset of symptoms was 3 days. DISCUSSION The results suggest that a low rate of endophthalmitis can be achieved by means of a protocol that includes the use of topical povidone-iodine, a REFERENCES 1. Peyman G, Lee P, Seal DV. Endophthalmitis: Diagnosis and Management. Taylor & Francis, London & New York: 2004: Kernt M, Kampik A. Endophthalmitis: Pathogenesis, clinical presentation, management,and perspective. Clin Ophthalmol 2010; 4: Avery RL, Bakri SJ, Blumenkrant MS,Brucker AJ, Cunningham ET Jr, D Amico DJ, Dugel PU, Flynn HW Jr, Freund KB, Haller JA, Jumper JM, Liebmann JM, McCannel CA, Mieler WF, Ta CN, sterile lid speculum, and topical anesthetic and postoperative application of topical antibiotics for seven days. It should be emphasized that this is the first study in B&H that documents the incidence of endophthalmitis after intravitreal application. It is known that bevacizumab in B&H is the most frequently used intravitreal anti-vascular endothelial growth factor due to very low price. In the last few months many efforts have been made to include ranibizumab and aflibercept to hospital list. A review of literature identified a number of reports of endophthalmitis rates after large series of intravitreal injections. The frequency of endophthalmitis after intravitreal anti VEGF reported in available scientific literature varies from 0.01% to 1.6% (13-18). We expect that with the increasing number of applications of anti VEGF, the overall number of endophthalmitis will increase as well in B&H in the near future. Despite the growing number of indications and agents for intravitreal injections, there is no consensus on peri-injection guidelines for the prophylaxis of endophthalmitis (19). Although endophthalmitis cannot be prevented in all cases, certain strategies must be proposed on the global level. In conclusion, endophthalmitis remains very a severe complication of intravitreal application of anti VEGF but infrequent. ACKNOWLEDGMENT The authors are grateful to patients who agreed to participate in this study. Also, authors thank professor Vahid Jusufović, Chief of Medical Retina Halida Basić and the entire team of Medical Retina at the University Clinical Center in Tuzla, Bosnia and Herzegovina. FUNDING No specific funding was received for this study. TRANSPARENCY DECELARATIONS Competing interest: none to declare. Williams GA. Intravitreal Injection Technique and Monitoring, Update Guidelines of an Expert Panel. Retina 2014; 34 (Suppl 12):S1-S Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY, Kim RY. MARINA study Group. Ranibizumab for neovascular age related macular degeneration. N Eng J Med 2006; 355: Regillo CD, Brown DM Abraham P, Yue H, Ianchulev T, Schneider S, Shamas N. Randomized, double-masked, sham-cotrolled trial of ranibizumab for 204

103 Terzić et al. Endophthalmitis after VEGF intravitreal application neovascular age-related macular degeneration: PIER Study year1. Am J Ophthalmol 2008; 145: Arevalo JF, Fromow-Guerra J, Quiroz-Mercado H, Sanchez JG, Wu L, Maia M, Berrocal MH, Solis- Vivanco A, Farah ME; Pan- American Collaborative Retina Study Group. Primary intravitreal bevacizumab (Avastin) for diabetic macular edema: result from the Pan-American Collaborative Retina Study Group at 6-month follow up. Ophthalmology 2007; 114: Rosenfeld PJ, Fung AE, Puliafito CA. Optical coherence tomography findings after an intravitreal injection of bevacizumab (Avastin) for macular edema from central retinal vein occlusion. Ophthalmic Surg Lasers Imaging 2005; 36: Ferrara N, Hillan KJ, Novotny W. Bevacizumab (Avastin), a humanized anti-vegf monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 2005; 333: Ramulu PY, Do DV, Corcoran KJ, Corcoran SL, Robin AL. Use of retinal procedures in medicare beneficiaries from 1997 to Arch Ophthalmol 2010; 128: Schwartz SG, Flynn HW Jr, Scott IU. Endophthalmitis after intravitreal injections. Expert Opin Pharmacother. 2009; 10; El-Ashray MI, Dhillon B. The article by Fintak et al. on the incidence of endophthalmitis related to intravitreal injections of bevacizumab and ranibizumab. Retina 2009; 29: Bhavsar AR, Googe JM, Jr, Stockdale CR, Bressler NM, Brucker AJ, Elman MJ, Glassman AR. Risk of Endophthalmitis After Intravitreal Drug Injection When Topical Antibiotics Are Not Required: The Diabetic Retinopathy Clinical Research Network Laser-Ranibizumab-Triamcinolone Clinical Trials. Arch Ophthalmol 2009; 127: Fung AE, Rosenfeld PJ, Reichel E. The International Intravitreal Bevacizumab Safety Survey: using the internet to assess drug safety worldwide. Br J Ophthalmol 2006; 90: Jonas JB, Spandau UH, Schlichtenbrede F. Shortterm complications of intravitreal injections of triamcinolone and bevacizumab. Eye (Lond) 2008; 22: Mason JO 3rd, White MF, Feist RM, Thomley ML, Albert MA, Persaud TO, Yunker JJ, Vail RS. Incidence of acute onset endophthalmitis following intravitreal bevacizumab (Avastin) injection. Retina 2008; 28: Diago T, McCannel CA, Bakri SJ, Pulido JS, Edwards AO, Pach JM. Infectious endophthalmitis after intravitreal injection of antiangiogenic agents. Retina 2009; 29: Jonas JB, Spandau UH, Rensch F, Von Baltz S, Schlichtenbrede F. Infectious and noninfectious endophthalmitis afterintravitreal bevacizumab. J Ocul Pharmacol Ther 2007; 23: Cavalcante LL, Cavalcante ML, Murray TG, Vigoda MM, Pina Y, Decatur CL, Davis RP, Olmos LC, Schefler AC, Parrott MB, alliman KJ, Flynn HW, Moshfeghi AA. Intravitreal injection analysis at the Bascom Palmer Eye Institute: evaluation of clinical indications for the treatment and incidence rates of endophthalmitis. Clin Ophthalmol 2010; 4: Englander M, Chen TC, Paschalis EI, Miller JW, Kim IK. Intravitreal injections at the Massachusetts Eye and Ear Infirmary: analysis of treatment indications and postinjection endophthalmitis rates. Br J Ophthalmol :460-5 Incidenca endoftalmitisa nakon intravitrealne primjene anti- VEGF terapije na Univerzitetsko kliničkom centru Tuzla Svjetlana Terzić, Adisa Pilavdžić, Amra Nadarević Vodenčarević Univerzitetsko klinički centar Tuzla, Klinika za očne bolesti, Tuzla, Bosna i Hercegovina SAŽETAK Cilj Utvrditi incidencu endoftalmitisa nakon intravitrealne aplikacije antivaskularnog endotelnog faktora rasta (anti-vegf) na Univerzitetsko kliničkom centru u Tuzli. Metode U ovoj retrospektivnoj studiji ukupno 986 intravitrealnih aplikacija bevacizumaba aplicirano je u periodu od januara do aprila godine na Univerzitetsko kliničkom centru u Tuzli (UKC Tuzla). Od januara godine ukupno je aplicirano 55 ranibizumaba dok je od oktobra broj apliciranih aflibercepta iznosio 60. Rezultati Tokom ovog perioda desila su se dva slučaja endoftalmitisa nakon aplikacije bevacizumaba, a nije se desio ni jedan slučaj nakon aplikacije ranibizumaba ili aflibercepta. Incidenca endoftalmitisa iznosila je 0,2%. Zaključak Rezultati indiciraju da se uz određene protokole može smanjiti incidenca endoftalmitisa. Ova studija je od velikog značaja jer se radi o prvoj studiji ovog tipa u Bosni i Hercegovini (BiH). Studijom su analizirani svi slučajevi endoftalmitisa nakon intravitrealne aplikacije anti-vegf terapije. U BiH je bevacizumab najčešće korišten anti-vegf zbog niske cijene. Ključne riječi: bevacizumab, aflibercept, ranibizumab, komplikacije nakon intravitrealne aplikacije 205

104 ORIGINAL ARTICLE Therapeutic efficacy and toxicity of bolus application of chemotherapy protocol in the treatment of metastatic colorectal cancer Ibrahim Šišić 1, Belma Pojskić 2, Alma Mekić-Abazović 1, Vladimir Kovčin 3 1 Department of Oncology, Hematology and Radiotherapy, 2 Department of Internal Diseases, Cantonal Hospital Zenica; Bosnia and Herzegovina, 3 Department of Oncology, Clinical Hospital Centre Bežanijska kosa Beograd, Serbia ABSTRACT Aim To compare efficacy and toxicity of bolus application of chemotherapy protocol, oxaliplatin, fluorouracil (bolus), leucovorin (folfox) between two groups of patients in the therapy of metastatic colorectal carcinoma (mcrc). Corresponding author: Šišić Ibrahim Department of Oncology, Hematology and Radiotherapy, Cantonal Hospital Zenica Crkvice 67, 7200 Zenica, Bosnia and Herzegovina Phone: ; Fax: ; sisicibrahim@yahoo.com Original submission: 29 December 2014; Revised submission: 10 February 2015; Accepted: 16 March doi: / Methods A total of 63 patients were treated for mcrc in the period January 2009 January 2010 at the Department of Oncology of the Cantonal Hospital Zenica, Bosnia and Herzegovina (first group, 30 patients) and at the Department of Oncology of the Clinical Hospital Centre Bežanijska kosa in Belgrade, Serbia, in the period January 2005 January 2006 (second group, 33 patients). The patients were treated according the same protocol, i.v. bolus infusion, but in different day intervals (D), 1, 8, 15/28 days or D1- D5/28 days, respectively. In all patients the following factors were analyzed: tumor response, overall survival (OS), progression free survival, hematological and non-hematological toxicity. Results Colon was the primary localization in almost two thirds of patients. There was no statistically significant difference between the groups according to the age, hematological and non-hematological toxicity, as well as in achieved OS. Progression free survival expressed in months was in average 5 months though with a large range between minimal and maximal survival time. Conclusion Both groups have shown equivalent efficacy to applied chemotherapy protocols. Overall survival in the two groups matched data from the literature. Further research should confirm success of the combination of chemotherapy protocols and their combination with the biological therapy. Key words: oxaliplatin, therapeutic response, overall survival, toxicity Med Glas (Zenica) 2015; 12(2):

105 Šišić et al. Efficacy and toxicity of bolus application INTRODUCTION It is estimated that every year colorectal cancer (CRC) affects about 1.2 million people and around 609,000 die as a consequence of CRC (1). The incidence increases with age (2,3). Elevated rates of incidence were estimated in European countries - Bosnia and Herzegovina (30 in men, 19 in women). Geographical patterns of mortality partially follow incidence. Estimated age-standardized rates (European standard) of cancer mortality by sex, cancer site and country 2012 in Bosnia and Herzegovina were 19.8 in men; 11.7 in women (4). Based on the data of the Cancer Register for Central Serbia it can be estimated that every year 4000 persons are affected by CRC in Serbia. Standardized incidence rate in Central Serbia is 33 per 100,000 in men and 19 per in women (5). Approximately 60% of diagnosed CRC cases develop metastatic disease. In the disease etiology three groups of risk factors can be mentioned: family history, life style and colon diseases (6). Among others, it is particularly important to mention familial adenomatous polyposis (FAP) in high-risk patients (7,8). It is believed that most colon cancers occur in a process of several levels or malignant transformation of adenoma through the process of activation of oncogenes and inactivation of tumor-suppressor genes, adenoma-carcinoma sequence (9). There are some opinions which negate so called malignant transformation of benign polyps, and it is believed that in most cases those are cancers from the very beginning - wolves in sheep s clothing (10). In cancer prevention stool tests performed once a year allow for early detection of cancer in 18-33%, sigmoidoscopy every five years in 34-55%, colonoscopy every 10 years in 75% of persons (11,12). Surgical treatment is a basis for the treatment of malignant diseases of the lower part of gastrointestinal system. A type of surgical treatment depends on tumor location (13,14 ). Possibilities of chemotherapy in patients with metastatic colorectal cancer (mcrc) are today promising thanks to oxaliplatin, irinotecan, capecitabine (5-fluorouracil+oxaliplatin, folfox, and 5-fluorouracil+irinotecan, folfiri) (15). Advantages of the selection of one of the these two protocols have been examined in a study by Tourgand (CER- COR study) according to which there is no significant difference in the overall survival regardless of selected therapy. However, there is a clear difference in the profile of toxicity, which means that the expected undesired differences are adjusted to age and potential comorbidities. Irinotecan proved to be safer in patients of older age (16,17). Based on results of OPTIMOX 1 study, suspension of the treatment is recommended in patients whose response to the treatment has been achieved or there is a stable disease, and after 6 or more cycles of the first-line treatment with FOLFOX protocol. In such patients a maintenance approach with Capecitabine or stop and go is advocated for, i.e. absence of therapy until metastases reach the previous size (OPTIMOX 2 study) (18). Studies examining three medicaments were published: combination of 5 FU, irinotecan and oxaliplatin (folfoxiri), which had some promising results though in certain younger populations of patients (19). Oral fluoropyrimidine (capecitabine) proved to be efficient and similar to 5FU/LV (5 fluorouracil /leucovorin), which is administered in a long-lasting iv and contributes to better quality of life (20). In the last ten years significant achievement has been made in the treatment of mcrc applying biological medicines. Target therapy needs to ensure simultaneous increase in efficiency and reduced toxicity of chemotherapy (21-23). In addition to numerous therapeutic protocols it is obvious that there is still no standardized therapy (24). Therefore, when it comes to the treatment of different subpopulations of patients with chemotherapy, it is necessary to select them according to numerous factors in order to achieve the highest possible number of patients to undergo curative R0 (clear margins post metastasectomy) liver resections or whose life will be prolonged to the maximum with significantly improved quality of life (24,25). The aim of this paper was primarily to compare efficacy of chemotherapy protocol of oxaliplatin, 5-fluorouracil (bolus), leucovorin (folfox) as a modified protocol in a three-week regimen (days), 1, 8, 15/every 28 days at the Oncology Department of the Cantonal Hospital of Zenica, with data of modified folfox protocol applied in five-day regimen of administering every 4 weeks at the Clinical Hospital Centre Bežanijska kosa in Belgrade. The secondary aim was to compare toxicity (hematological and non-hematological) of these two modes of bolus application of chemotherapy protocol (folfox protocol) in the 207

106 Medicinski Glasnik, Volume 12, Number 2, August 2015 treatment of metastatic colorectal cancer (mcrc). The parameters followed in both groups of patients were: overall therapeutic response, time to progression of the disease, overall survival (OS), and toxicity per number of chemotherapy cycles. PATIENTS AND METHODS This retrospective study included 63 patients: first (research) group (30), and second (control) group (33) in the period of one year. The study was conducted at the Cantonal Hospital Zenica in the period January 2009 January 2010 (30 patients, research group), and at the Clinical Hospital Centre Bežanijska kosa in Belgrade in the period between January 2005 and January 2006 (33 patients, control group). The study included patients who had a verified diagnosis of metastatic colorectal cancer, histologically identified as invasive adenocarcinoma. The research group was treated at the Oncology Department of the Cantonal Hospital Zenica. The control group consisted of patients with same pathohistological diagnosis, e.g. metastatic colorectal carcinoma with good performance status, who were treated at the Oncology Department of the Clinical Hospital Centre Bežanijska kosa in Belgrade, with the same chemotherapy protocol (folfox protocol) as bolus infusion, but with different regimens, i.e. administration time intervals. In all patients the survival was calculated from the date of the first chemotherapy cycle until the date of death as a result of any cause, and if this information was not available, until the date of the last control examination. All collected data were analyzed applying methods of descriptive and analytical statistics: χ2 test, T-test, U test, normal distribution test, Wilcoxon Signed Ranks test, Mann-Whitney, Spearman rank correlation were used for statistical analysis. The T-test was used to access the average patients age, χ2 test was used to analyze the distribution of patients to subsets according to therapeutic toxicity, Mann-Whitney to analyze the distribution of patients to subsets according to therapeutic response, time to progression, and overall survival. RESULTS The study included 63 patients of average age of 60 years. The youngest patient was 34 years old, while the oldest one was 74 years old. There is no statistically significant difference between the groups according to the age (p=0.269). Average difference between groups was 2.6 years (Table 1). Colon was the primary localization in almost two thirds of patients. Susceptibility to colon localization was noticed in 42 (67.7%), while 20 (32.3%) patients had rectal cancer. As far as gender is concerned, there was no significant difference in the distribution of primary localization of cancer (p=1.000) Table 1. Average age, median and variability of years of age in research and control group of patients Group No (%) of Arithmetic SD Median Minimumum Maxi- patients mean Research 30 (48) Control 33 (52) Total 63 (100) Analyzing distribution of patients per groups in relation to toxicity by dividing them to those who had or had no therapy-related toxicity, there was no statistically significant difference between the groups (p=0.424) (Table 2). Table 2. Distribution of patients per groups in relation to toxicity of the chemotherapy No (%) of patients Group of patients Toxicity of therapy NO YES Total Research 11 (36.7) 19 (63.3) 30 (100) Control 9 (27.3) 24 (72.7) 33 (100) Total 20 (31.7) 43 (68.3) 63 (100) In terms of therapeutic response, out of the total of 63 patients for whom data is available, only nine (14.8%) patients had partial remission (PR), (five patients in the examined and four patients in the control group). One patient in the examined group had complete remission, while the highest number of patients, 40 (65.6%; 22 in the examined and 18 in the control group) had the progression of the disease (PD) mainly after the third cycle. There was no statistically significant difference between the groups according to therapeutic response (p=0.431) (Table 3). Table 3. Distribution of patients per groups in relation to therapeutic response No (%) of patients Group of patients Therapeutic response PD SD PR CR Total Research 22 (73.3) 2 (6.7) 5 (16.7) 1 (3.3) 30 (100) Control 18 (58.1) 9 (29) 4 (12.9) 0 (0) 31 (100) Total 40 (65.6) 11 (18.0) 9 (14.8) 1 (1.6) 61 (100) PD, disease progression; SD, stable disease; PR, partial regression; CR, complete response 208

107 Šišić et al. Efficacy and toxicity of bolus application The applied therapy protocol assured stable disease, in terms of response, only to 11 (18%) patients (p=0.431). Duration of the response was 5 months in average, with a large range between the minimum and maximum. There is no statistically significant difference between the groups according to time to progression (p=0.880) (Table 4). Table 4. Arithmetic mean, SD, median and variability of time to disease progression Group of No (%) of Arithmetic SD Median Minimumum Maxi- patients patients mean Research 30 (52) Control 28 (48) Total 58 (100) The overall survival of patients in this study was 23 months (in average with standard deviation of 14.5). Due to high standard deviation, the best indicator of overall survival is median survival and it was 20 months (Table 5). Three patients had overall survival more than five years. There was no statistically significant difference in achieved overall survival (OS) in the two groups (p=0.840). Time to progression (TTP) was 5 months in average, though with a large range between minimal and maximal survival time. Table 5. Arithmetic mean, SD, median and variability of patients overall survival Group of No (%) of Arithmetic SD Median Minimumum Maxi- patients patients mean Research 30 (49) Control 31(51) Total 61 (100) DISCUSSION Chemotherapy protocols in the treatment of CRC are selected according to the National Comprehensive Cancer Network (NCCN) guidelines (26,27). With introduction of biological therapy (bevacizumab, cetuximab and panitumumab) with the chemotherapy protocols median survival higher than two years was achieved. Without treatment patients with metastatic colorectal cancer (mcrc) live in average for five to six months (15). Usual protocols for the treatment of mcrc are given in bolus. Current standard protocol includes administration of continuous infusion in the period of 48 hours. Such application achieves better therapeutic effect (22:14 %), longer median survival (12.1:11.3 months; p=0.04) and reduction of myelotoxicity (4:31%) (15). The patients of the first group, in the Cantonal Hospital of Zenica, received folfox bolus protocol in the three-week regimen: day 1 (D1); day 8 (D8); day 15 (D15) / every 28 days, and the patients of the second group at the Clinical Hospital Centre Bežanijska Kosa, Belgrade, received folfox-bolus protocol in the five-day regimen, D1-D5 /every 28 days. Protocol modification, which means chemotherapy administration via bolus in different time frames (D1- D5 e.g., D1, 8, 15, every 28 days) in analyzed groups vs standardized continuous protocols over 48 hours administration time, was applied to enable the application administration in the conditions of daily hospital with patients going home every day after the therapy, ambulatory patients or daily clinic patients, and it is very comfortable for patients. Analyzing groups examined with stage IV disease (metastatic disease) it is crucial to set the implementation of systemic measures of primary and secondary prevention as a basic task of our health care system (28,29). The patients in the examined sample received 4 cycles of chemotherapy in average. Like any other chemotherapy, folfox therapeutic protocol also causes various side-effects. The study results indicate that the majority of patients had therapeutic response within 6 months, which is statistically significant. Comparing the data from the literature it could be concluded that they match the overall survival in this study. Multidisciplinary decision and individualized approach are the main principles when treating this heterogeneous group of patients. The study supports administration of both protocols in clinical practice, but when taking into consideration lowing of the costs and less patients visits, three-day administration regimen, can be considered preferred. It is the major conclusion of the study that there is a crucial need for implementing prevention methods (primary and secondary) for early screening and detection of colorectal carcinoma, which would bring a long term therapeutic effect when treating colorectal carcinoma, but would also lower the costs. Further research would need to be directed towards combining chemotherapy medications 209

108 Medicinski Glasnik, Volume 12, Number 2, August 2015 and different protocols with biological medicines, which could result in even higher overall survival and decrease in undesired effects. TRANSPARENCY DECLARATION Competing interests: None to declare. FUNDING No specific funding was received for this study. REFERENCES 1. Benson AB. Epidemiology, disease progression and economic burden of colorectum cancer. JMCP 2007; 13: Boyle P, Levin B. World cancer report Lyon: International Agency for Research on Cancer, Vrdoljak E, Wojtukeiwicz MZ, Pienkowski T, Bodoky G, Berzinec P, Finek J, Todorović V, Borojević N, Croitoru A. Cancer epidemiology in Central and South Eastern European countries. CMJ 2011; 52: Fearlay J, Steliarova-Foucher E, Loret-Tieulent J, Rosso S, Coebergh JWW, Comber H, Forman D, Bray F. Cancer incidence and mortality patterns in Europe: Estimates for 40 countries in EJC 2013; 49: Institut za javno zdravlje Srbije Jovan Jovanović Batut. Incidencija i mortalitet od raka u centralnoj Srbiji Beograd: Insititut za javno zdravlje, Eduard V, Mirko Š, Zvonko K, Marija P, Damir P, Damir G, Zdneko K. Klinička onkologija. Zagreb: Medicinska naklada, Ashan H, Neugut AL, Garbovski GC, Jacobson JS, Forde KA, Treat MR, Waye JD. Family history of colorectal adenomatous polyps and increased risk for colorectal cancer. Ann intern Med 1998; 129: Fuchs CS, Giovannucci EL, Colditz GA, Hunter DJ, Speizer FE, Willett WC. A prospective study of family history and the risk of colorectal cancer. N Engl J Med 1994; 331: Winawer SJ. Natural history of colorectal cancer. Am J Med 1999; 103:3S-6S 10. Koretz RL. Malignat polyp: are they sheep in wolves, clothing? Ann Intern Med 1993; 118: Winawer S, Fletcher R, Rex D, Bond J, Burt R, Ferrucci J, Ganiatis T, Levin T, Woolf S, Johnson D, Kirk L, Litin S, Simmang C. Colorectal cancer screening and surveillance: clinical guidelines and rationale-update based on now evidence. Gastroenterology 2003; 124: U.S. Preventive Services Task Force. Screening for colorectal cancer: U.S. Preventive Services Task Force Recommendation Statement. Ann Inter Med 2008; 149: Cochen A.M. Surgical consideration in patients with cancer of the colon and rectum. Semin Oncol 1991; 18: Fazio VW,Church JM, Delaney CP. Current Therapy Colon and Rectal Surgery. 2 nd ed. Philadelphia: Elsevier Mosby, 2005; Dobrila-Dintijana R, Bagić Ž, Štimac D. Kemoterapija kolorektalnog karcinoma. Medix 2008; Tournigand C, Andre T, Achille E, Liedo G, Flesh M, Mery-Mignard D, Quinaux E, Couteau C, Buyse M, Ganem G, Landi B, Colin P, Louvet C, de Gramont A. FOLFIRI followed by FOLFOX6 or the reverse seguencein advanced colorectal cancer: a randomized GERCOR study. J Clin Oncol 2004; 22: Goldberg RM, Morton R, Sargent D, Fuchs C, Ramanathan R, Williamson S, Findlay BP. Oxaliplatin (oxal) or CPT Fluorouracil/Leucovorin or Oxal +CPT-11in advanced colorectal cancer: Initial toxicity and response data from a GI Intergroup study. Pro Am Soc Clin Oncol 2002; 21: Maindrault-Goebel F, Leido G, Chibaudel B, Mineur T, Andre M, Bennamoun M, Mabro P, Artru C, Louvet C, de Gramont A. OPTIMOX2, a large randomized phase II study of maintenance therapy of chemotherapy-free intervals(cfi) after FOLFOX in patints with metastatic colorecral cancer (MRC). J Clin Oncol 2006; 24:147s. 19. Souglakos J, N. Androulakis, K Syrigos, A Polyzos, N Ziras, A Athanasiadis, S Kakolyris, S Tsousis, Ch Kouroussis, L Vamvakas, A Kakykaki, G Samonis, D Mavroudis and V Georgoulias. FOLFOXIRI (folin acid, 5- fluorouracil, oxaliplatin and irinotecan) vs FOLFIRI (folin acid,5-fluorouracil and irinotecan) as first- line treatment in metastatic colorectal cancer (MCC): a multicentre randomized III trail from the Hellenic Oncology Research Group (HORG), Br J Cancer 2006; 94: Kovčin V, Ješić R, Krivokapić Z, Andrić Z, Pavlović A. Xeloda as first-line chemotherapy of metastatic colorectal cancer-our expirence. Arch Oncology 2002; 10: Popov I, Tarabar D, Jovanović D, Kovčin V, Radić S, Micev M, Petrović Z, Manojlović N, Andrić Z, Dagovič A, Kukić B, Radišević-Jelić LJ, Kecmanović D, Josifovski J, Jezdić S, Milović M, Milošević N, Stanković J, Borojević N, Ćeranić M, Pavlov M, Stojanović S, Stanković V, Kežić I. Efficacy and safety of bevacizumab in combination with oxaliplatin, irinotecan and fluoropyrimidine-based therapy in advanced colorectal cancer. Arch Oncol 2007; 15: Saltz LB, Clarke S, Diaz-Rubio E, Scheithauer W, Figer A, Wong R, Koski S, Lichinitser M, Tsai-Shen Y, Rivera F, Couture F, Sirzen F, Cassidy J. Bevacizumab in combination with oxaliplatin based chemotherapy as first-line therapy in metastatic colorectal cancer: a randomized phase III study. J Clin Oncol 2008; 26:

109 Šišić et al. Efficacy and toxicity of bolus application 23. Cutsem EV, Claus-Henning K, Hitre E, Zaluski J, Chung-Rong CC, Makhson A, Geert D Haens G, Pinter T, Lim R, Bodoky G, Roh JK, Folprecht G, Ruff P, Stroh C, Tejpar S, Schlichting M, Nippgen J, Rougier P. Cetuximab and Chemotherapy as Initial Treatment for Metastatic Colorectal Cancer. N Engl J Med 2009; 360: Krivokapić Z. Karcinom rektuma. Beograd: Zavod za udžbenike, Kopetz S, Chang GJ, Overman MJ, Enq C, Sargent DJ, Lasron DW, Grothey A, Vauthe JN, Nagorney DM, McWilliams RR. Improved survival in metastatic colorectal cancer is associated with adoption of hepatic resection and improved chemotherapy. J Clin Oncol 2009; 27: Benson AB, Venok AP, Bekall-Saab T, Chan E, Yi- Jen Ch, Cooper HS, Engstrom PF, Enzinger PC, Fenton MJ, Fuchs CS, Grem JL, Grothey A, Hochster GS, Hunt S, Kamel A, Kirilcuk N, Leong LA, Lin E, Messersmith WA, Mulcahy MF, Murphy JD, Nurkin S, Rohren E, Ryan AP, Saltz L, Sharma S, Shibata D, Skibber JM, Sofocleous CT, Stoffel ES, Stotsky-Himelfarb E, Willett CG, Freedman-Cass D. National Comprehensive Cancer Network. (NCCN) Clinical Practice Gudelines in Oncology (NCCN Guidelines). Colon Cancer. 20 th Annual Ed. V.2. New York: Cold Spring Publishing, www. nccn.org/professionals/physician_gls/pdf/colon.pdf. (11 March.2015) 27. Benson AB, Venok AP, Bekall-Saab T, Chan E, Yi- Jen Ch, Cooper HS, Engstrom PF, Enzinger PC, Fenton MJ, Fuchs CS, Grem JL, Grothey A, Hochster GS, Hunt S, Kamel A, Kirilcuk N, Leong LA, Lin E, Messersmith WA, Mulcahy MF, Murphy JD, Nurkin S, Rohren E, Ryan AP, Saltz L, Sharma S, Shibata D, Skibber JM, Sofocleous CT, Stoffel ES, Stotsky-Himelfarb E, Willett CG, Freedman-Cass D. National Comprehensive Cancer Network. Upisati puni naziv ustanove-autora (NCCN). Clinical Practice Gudelines in Oncology. Rectal Cancer. 20 th Annual Ed. V.2. New York: Cold Spring Publishing, pdf/rectal.pdf.(11 March 2015) 28. De Vita VT, Lawrence TS, Rosenberg SA. Cancer Principels & Practice of Oncology. 8 th Ed. Philadelphia: Lippincott Williams & Wilkins, Rex DK, Johnson DA, Anderson JC, Schoenfeld PS, Burke CA, Inadomi JM. American College of Gastroenterology Guidelines for colorectal cancer screening Am J Gastroenterology 2009; 104: Terapijska efikasnost i toksičnost bolusnih primjena hemioterapijskog protokola u terapiji metastatskog kolorektalnog karcinoma Ibrahim Šišić 1, Belma Pojskić 2, Alma Mekić-Abazović 1, Vladimir Kovčin 3 1 Služba za onkologiju, hematologiju i radioterapiju, 2 Služba za unutrašnje bolesti; Kantonalna bolnica Zenica, Bosna i Hercegovina; 3 Odeljenje za onkologiju, Kliničko-bolnički centar Bežanijska kosa, Beograd, Srbija. SAŽETAK Cilj Uporediti terapijsku efikasnost i toksičnost bolusnih primjena hemioterapijskog protokola, oxaliplatine, fluorouracil (bolus), leukovorin (folfox), između dvije grupe bolesnika u terapiji metastatskog kolorektalnog karcinoma. Metode Ukupno 63 bolesnika liječena su od metastatskog kolorektalnog karcinoma, u periodu od januara do januara godine, na Onkološkom odjeljenju Kantonalne bolnice Zenica, Bosna i Hercegovina (prva grupa od 30 pacijenata ) i na Onkološkom odjeljenju Kliničko-bolničkog centra Bežanijska kosa u Beogradu, Srbija, u periodu od januara do januara godine (druga grupa od 33 pacijenta). Pacijenti su bili tretirani istim hemioterapijskim protokolom, i.v. bolus infuzije, ali u različitim vremenskim intervalima, D1, 8, 15/28, odnosno D1-D5/28 dana. Kod svih pacijenata analizirani su terapijski odgovor, ukupno preživljavanje (OS), vrijeme do progresije bolesti, kao i toksičnost. Rezultati Primarna lokalizacija u skoro dvije trećine pacijenata bio je kolon. Nisu ustanovljene statistički značajne razlike između skupina prema dobi, u hematološkoj i nehematološkoj toksičnosti, kao ni u ukupnom preživljavanju. Vrijeme do progresije bolesti u mjesecima bilo je u prosjeku pet mjeseci, ali s velikim rasponom između minimalnog i maksimalnog. Zaključak Ustanovljena je podjednaka efikasnost hemioterapije u obje grupe bolesnika. Ukupno preživljavanje u dvije grupe bilo je podudarno s podacima iz literature. Dalja istraživanja trebala bi potvrditi uspješnost kombinacije hemioterapijskih protokola i njihove kombinacije s biološkom terapijom. Ključne riječi: oxaliplatin, terapijski odgovor, ukupno preživljavanje, toksičnost 211

110 ORIGINAL ARTICLE Differences in body mass index and height factors between men with and without varicocele Hamid Shafi 1, Mouloud Agajani Delavar 2 1 Department of Surgery, 2 Department of Midwifery; Fatemezahra Infertility and Reproductive Health Research Center, Babol University of Medical Sciences, Babol, Islamic Republic of Iran ABSTRACT Aim Despite many studies published in recent years concerning the relationship between demographic factors and varicocele, this issue remains controversial. The aim of this study was to identify a possible influence of body mass index (BMI) and height on occurrence varicocele in men. Methods In a case-control study 153 patients aged years from 2004 to 20014, with moderate and sever varicocele were studied. The BMI and height of the 153 patients with varicocele were compared with 250 men who had no varicocele as a control group. Corresponding author: Mouloud Agajani Delavar Department of Midwifery, Infertility and Reproductive Health Research Center of Babol, University of Medical Sciences, P.O. Box: , Babol, Mazandaran, Iran Tel: ; Fax: ; moloodaghajani@yahoo.com Results After the adjustment for socio-demographic factors, the risk of varicocele for obese men was lower than for overweight and normal men (OR= 0.38, 95% CI= 0.17, 0.85). The adjusted OR for varicocele in taller men was higher than in those with low height (OR= 3.42, 95% CI= 1.34, 8.72), and moderate height (OR=2.68, 95% CI= 1.12, 6.46). Conclusion The results of this study indicated that tall men and non-obese men may be at higher risk of varicocele, therefore counseling and evaluation of the men at high risk of varicocele may be of benefit for reduced infertility. Key words: pampiniform plexus, spermatic vein, anthropometric parameters Original submission: 14 October 2014; Revised submission: 09 February 2015; Accepted: 01 June doi: / Med Glas (Zenica) 2015; 12(2):

111 Shafi et al. BMI, height, and varicocele INTRODUCTION Varicocele is a dilatation of the testicular veins and swelling of network of veins from the testicles within spermatic cord (1). It is likely that varicocele can cause infertility if it associated with abnormal semen analysis (2,3). Estimated prevalence varies considerably in general population and infertile men (4). It is important to note that the prevalence of varicocele has increased tremendously over the past decades (5,6). The exact mechanism of varicocele is unclear. However, the interaction of increased pressure in the veins of the testicles and its venous drainage, as well as genetic factors suggested to be the factors that contribute to its development (6, 7). Several studies have shown that varicocele may be decreased in overweight and obese men and increased with taller men (5, 7-9). Similarly, obesity may play a role in detection of varicocele in obese men (10). In contrast, a study showed that the risk of varicocele is associated not only with low body mass index (BMI), but also with high BMI (11). Varicocele is the most common cause of seminal abnormalities (12). However, varicocele is not associated with infertility despite seminal abnormalities (13), but progressive infertility as varicocele increases surgical repairs in Iranian population. It is critical to identify association between varicocele with several parameters such as BMI and height in men. PATIENTS AND METHODS The study design was a case-control study. It was performed on 153 patients with moderate and severe varicocele who were referred to the Outpatient Urology Department for varicocele repair in the period Two hundred and fifty patients who had no varicocele were randomly selected, matched to cases based on age as the control group. The men with clinical unilateral or bilateral varicocele were detected in the outpatient clinic by the same investigator during physical examination in the upright position, and confirmed by Doppler ultrasongraphy of the scrotum in a warm room (>23 C). The men with a history of varicocelectomy and / or inguinal hernia surgery, hydrocele, absence of any testicular for any reason, any musculoskeletal disease or deformity were excluded from the study. Weight was recorded using digital scales, with the subjects minimally without shoes and with a tape measure. The body mass index was calculated using the formula: BMI=weight (kg)/ height 2 (m). According to the National Institute of Health and Clinical Excellence (14), a patient is placed in one of the three BMI categories (normal weight - less than 25 kg/m 2 ), overweight ( kg/m 2 ), and obese ( 30 kg/m 2 ). Height was measured with a tape measure. The height of all subjects was categorized by tertiles. The frequency of varicocele in each tertile category was compared. This study was approved by the Ethical Committee of Babol University of Medical Sciences. Informed written consent was obtained from all eligible subjects (>18 years). Descriptive statistics were used to describe baseline demographics. To determine association between categorical BMI and height with varicocele, varicocele was considered as a dependent variable for logistic factors included height, education, BMI, residency and occupation of the men were adjusted as confounders. p< 0.05 was considered statistically significant. RESULTS The mean age of the men with varicocele was 26.7±4.9, and for that one without varicocele it was 26.1±6.0 years, ranged between years (p=0.309). All participants had mean height and mean weight 174.4±7.7 cm and 74.1±12.3 kg, respectively. The mean value for height and weight of the men with varicocele was 175.5±6.5 cm and 73.3±12.1 kg, respectively. The mean of height and weight of these without varicocele was 173.8±8.2 cm and 74.5±12.3 kg, respectively. The mean BMI of the all participants was 24.3±3.6 kg/m 2. The patients with varicocele had significantly lower BMI (0.014) than the men without varicocele (Table 1). 213

112 Medicinski Glasnik, Volume 12, Number 2, August 2015 Table 1. Characteristics of subjects according to varicocele Variables Total (n=403) Patients without varicocele (n=250) Patients with varicocele (n=153) Age (Mean±SD) 26.4± ± ± Height (Mean±SD) 174.4± ± ± Weight (Mean±SD) 74.1± ± ± Body mass index (Mean±SD) 24.3± ± ± Education (No/%) Illiterate (No/%) 9 (2.2) 7 (2.8) 2 (1.3) < (41.4) 60 (39.2) 107 (42.8) (33.7) 41 (26.8) 95 (38.0) (22.6) 50 (32.7) 41 (16.4) Residency (No/%) Urban 274 (68.0) 182 (27.8) 92 (60.1) Rural 129 (32.0) 68 (27.2) 61 (39.9) Occupations (No/%) Office employees 30 (7.4) 25 (10.0) 5 (3.3) Industrial/ construction workers 105 (26.1) 31 (12.4) 74 (48.4) Drivers 20 (5.0) 14 (5.6) 6 (3.9) Farmers 26 (6.5) 10 (4.0) 16 (10.5) Business 155 (38.5) 129 (51.6) 26 (17.0) The mean of height and weight according to each varicocele category is shown in Table 2. Statistically significant differences between BMI with each grade of varicocele have been found. There was no statistically significant difference between height and severity of varicocele. Table 2. The mean values of body mass index (BMI) and height according to each varicocele category Variables Grade III Grade I/II Non-varicocele p BMI (kg/m 2 ) 23.5± ± ±3.8 <0.05 Height (cm) 176.5± ± ±8.21 >0.05 The adjusted OR for varicocele in taller men was significantly higher than in men with low height (OR= 3.42; 95% CI= 1.34, 8.72), and moderate height (OR=2.68; 95% CI= 1.12, 6.46). The risk of varicocele for obese men was found to be lower than in man with overweight and normal BMI (OR= 0.38; 95% CI= 0.167, 0.852) (Table 3). Table 3. Adjusted ratio (OR) for varicocele according to body mass index (BMI) and height of subjects Variables Adjusted OR* 95% Confidence Interval BMI (kg/m 2 ) BMI (kg/m 2 ) Obese , Overweigh , Normal 1.00 Height (cm) , , < *Adjusted for confounder were height, BMI, education, occupation and residency; Obese: BMI 30 kg/m 2 ; Overweight: BMI= kg/m 2 p DISCUSSION Numerous researchers have assessed the relationship between varicocele and BMI. It is suggested that in obese men excess fat around the renal vein provides a cushion protecting against the nutcracker phenomenon (15-17). But yet, relationship between varicocele and BMI is controversial. The occurrence of varicocele may be decreased with increasing BMI (5,10,15,18). The results of this study support these findings. In contrast, some researchers have shown no significant differences between varicocele and BMI (19-20). In addition, Baek et al. (2011) showed the men with varicocele had a lower BMI (11). Also, in our study, men with severe varicocele had the highest mean of BMI compared with non varicocele men, which is in agreement with two studies, where authors reported that severity of varicocele inversely correlated with obesity (8, 16). Since it is well known that palpation of scrotum in obese men is difficult, it may lead to decreased diagnosis of varicocele (11). It could not be discounted that low grade varicocele may have been missed on physical examination (21). More research is needed to define a role of BMI and pathogenesis of varicocele. Our finding showed that men with varicocele were significantly taller than the men without varicocele. This finding is consistent with several studies (7, 22). Some studies emphasized that the greater height increases hydrostatic pressure in the spermatic vein, which may have a role in the valve mechanisms in the veins and lead to development of varicocele (9, 16). Several studies reported that higher height was not associated with higher risk of varicocele (22,1). Results of our study support this concept that height directly affected the prevalence of varicocele. Nevertheless, there is no direct evidence to show that valve mechanisms effect is related to the presence of varicocele. Larger control studies may possibly clarify the role of height in the pathogenesis of varicocele. A limitation of this study is that the study population was selected from only one outpatient clinic, Although there is another urology clinic in Babol, involving of all urology clinics in this study was difficult to achieve. In addition, all men in the control group were selected in the same outpatient clinic, and therefore, the results of the study cannot be considered as a representative of the general Ira- 214

113 Shafi et al. BMI, height, and varicocele nian male population. Another limitation is in the number of obese patients because a convenience sample was used instead of population based random sample. Another drawback of this study was related to data collection; unavailability of some information that can affect varicocele in men. The findings were nearly comparable with the findings in other countries. Data from the present study have shown that males with varicocele had lower BMI and higher height than men without varicocele. Counseling and evaluation of men at high risk of varicocele, especially for couples attended to infertility center, could be beneficial. ACKNOWLEDGEMENTS The authors acknowledge the assistance of Iranian men for their participation in this study, and the assistance Maryam Rahimi in the sampling. FUNDING This research was supported by the Infertility and Reproductive Health Research center Babol University of Medical Sciences. TRANSPARENCY DECLARATIONS Conflict of interest: none to declare. REFERENCES 1. Soylemez H, Atar M, Ali Sancaktutar A, Bozkurt Y, Penbegul N. Varicocele among healthy young men in Turkey; prevalence and relationship with body mass index. Int Braz J Urol 2012; 38: Bozhedomov VA, Lipatova NA, Rokhlikov IM, Alexeev RA, Ushakova IV, Sukhikh GT. Male fertility and varicocoele: role of immune factors. Andrology 2014; 2: Kwak N, Siegel D. Imaging and interventional therapy for varicoceles. Curr Urol Rep 2014; 15: Agarwal A, Sharma RK, Desai NR, Prabakaran S, Tavares A, Sabanegh E. Role of oxidative stress in pathogenesis of varicocele and infertility. Urology 2009; 73: Rais A, Zarka S, Derazne E, Tzur D, Calderon-Margalit R, Davidovitch N, Afek A, Carel R, Levine H. Varicocoele among Israeli adolescent males: time trends and association with body mass index. Andrology 2013; 1: Kumanov P, Robeva RN, Tomova A. Adolescent varicocele: who is at risk? Pediatrics 2008; 121:e Gokce A, Demirtas A, Ozturk A, Sahin N, Ekmekcioglu O. Association of left varicocoele with height, body mass index and sperm counts in infertile men. Andrology 2013; 1: Doğantekin E, Görgel SN, Şahin E, Girgin C. Relationship between varicocele and anthropometric indices in infertile population. Dicle Medical Journal 2014; 41: Hassanzadeh K, Yavari-Kia P, Soleymanpour H, Ebrahimpour N, Alikhan H. Effect of body mass index and prevalence of varicocele. Pak J Biol Sci 2011; 14: Chen SS, Huang WJ. Differences in biochemical markers and body mass index between patients with and without varicocele. J Chin Med Assoc 2010; 73: Baek M, Park SW, Moon KH, Chang YS, Jeong HJ, Lee SW, Han SW, Kim YS; Korean Society of Pediatric Urology. Nationwide survey to evaluate the prevalence of varicoceles in South Korean middle school boys: a population based study. Int J Urol 2011; 18: Pasqualotto FF, Pasqualotto EB, Sobreiro BP, Hallak J, Medeiros F, Lucon AM. Clinical diagnosis in men undergoing infertility investigation in a university hospital. Urol Int 2006; 76: Hauser R, Paz G, Botchan A, Yogev L, Yavetz H. Varicocele: effect on sperm functions. Hum Reprod Update 2001; 7: National Clinical Guideline Centre (UK). Obesity: Identification, Assessment and Management of Overweight and Obesity in Children, Young People and Adults: Partial Update of CG ; Handel LN, Shetty R, Sigman M. The relationship between varicoceles and obesity. J Urol 2006; 176: Tsao CW, Hsu CY, Chou YC, Wu ST, Sun GH, Yu DS, Fan PL, Chen HI, Chang SY, Cha TL. The relationship between varicoceles and obesity in a young adult population. Int J Androl 2009; 32: Nielsen ME, Zderic S, Freedland SJ, Jarow JP. Insight on pathogenesis of varicoceles: relationship of varicocele and body mass index. Urology 2006; 68: Al-Ali BM, Shamloul R, Pichler M, Augustin H, Pummer K. Clinical and laboratory profiles of a large cohort of patients with different grades of varicocele. Cent European J Urol 2013; 66: Kilic S, Aksoy Y, Sincer I, Oguz F, Erdil N, Yetkin E. Cardiovascular evaluation of young patients with varicocele. Fertil Steril 2007; 88: Delaney DP, Carr MC, Kolon TF, Snyder HM 3rd, Zderic SA. The physical characteristics of young males with varicocele. BJU Int 2004; 94: Chanc Walters R, Marguet CG, Crain DS. Lower prevalence of varicoceles in obese patients found on routine scrotal ultrasound. J Urol 2012; 187: May M, Taymoorian K, Beutner S, Helke C, Braun KP, Lein M, Roigas J, Hoschke B. Body size and weight as predisposing factors in varicocele. Scand J Urol Nephrol 2006; 40: Maghraby HA. Laparoscopic varicocelectomy for painful varicoceles: merits and outcomes. J Endourol 2002; 16:

114 ORIGINAL ARTICLE Genetic polymorphisms variants in interleukin-6 and interleukin- 1beta patients with obstructive sleep apnea syndrome in East Northern Turkey Ilhami Gok 1, Nergiz Huseyinoglu 2, Dogan Ilhan 3 1 Department of Bioengineering, Faculty of Engineering and Architecture, 2 Departments of Neurology, School of Medicine, 3 Departments of Molecular Biology and Genetics, Faculty of Science and Literature; Kafkas University, Kars, Turkey ABSTRACT Aim To investigate the relationship of IL-1β and IL-6 cytokine gene polymorphisms with obstructive sleep apnea syndrome (OSAS) in 61 patients admitted to the neurology clinic in Kafkas University Hospital with insomnia problem who were diagnosed with OSAS in sleeping labs, and 80 healthy subjects not associated with the syndrome. Corresponding author: Ilhami Gök Department of Bioengineering, Faculty of Engineering and Architecture, Kafkas University 36100, Kars, Turkey Phone: ; Fax: ; E.mail: dnzgoki@gmail.com Original submission: 12 January 2015; Revised submission: 13 March 2015; Accepted: 23 March doi: / Methods Blood samples were taken to isolate DNA from patients diagnosed with OSAS based on polysomnography results and healthy controls. DNA amplification of the genes was performed with PCR. Amplification products were cut with the restriction enzymes in order to determine IL-1 gene (TaqI) and IL-6 gene (Lwel) polymorphisms. The cut DNA fragments were carried out in agarose gel electrophoresis, and RFLP analysis was performed by utilizing the images with gel imaging system. PCR products were sequenced with an Applied Biosystems Automated Sequencer. Results Polymorphic changes were observed for IL-1β gene in 26 of 62 patients (41.9%), and 16 of the 80 (25.8%) in the control group. The incidence of polymorphic changes in IL-6 gene was in seen in seven (of the 62 patients) (11.3%), and in the 16 (20%) controls. Conclusion The findings on the genomic level in OSAS may provide an important contribution to diagnosis of obstructive sleep apnea syndrome in clinical practice, as well as it helps to obtain the results easily about environmental and genetic interaction of OSAS patients. Key words: OSAS, cytokine genes, RFLP, Turkey Med Glas (Zenica) 2015; 12(2):

115 Gok et al. Gene polymorphisms and sleep apnoea syndrome INTRODUCTION Sleep is an essential part of health and sleepbreathing disorders can cause serious health problems, economic losses both social and personal. The most common type of sleep apnea is defined as obstructive disorders (1). Obstructive sleep apnea syndrome (OSAS) disease constitutes a risk in people with certain anatomical features such as obesity, advanced age, gender and short neck (2). Daytime sleepiness, snoring, airway obstruction are the main findings of apnea (3). Traffic accident rate is high with these patients because of excessive daytime sleepiness (2). Sleep apnea is a risk for the development of many systemic diseases and is involved in the etiopathogenesis of various cardiovascular and disease progression (3). Obstructive sleep apnea syndrome and obesity are disorders common in individuals (2-3). Disorders associated with obesity have become some of the most serious social problems that triggered the formation of OSAS in recent years. Obstructive sleep apnea syndrome is a disease characterized in the upper respiratory tract in adults and in middle-aged individuals; it often leads to hypoxia and affects the sympathetic nervous system, metabolic reactions may also lead to an increase in blood pressure (3-4). As the obesity is a multifactorial disease, a significant relationship between genetic factors, obesity and OSAS has been defined (5). In OSAS, the reason of the airway obstruction is that the soft tissue slumps down the trachea during sleep. In each apnea event, enough oxygen does not get to the brain and sleep of persons with syndrome is broken down due to suffocation, and sleep is of poor quality and fragmented (6). Obstructive sleep apnea syndrome, in general, is common in overweight adults, in patients with diabetes. Risk factors include male gender, obesity, and age. Sleep apnea may be seen at any age, or even in children (6-7). Cytokine genes of interleukin-1β (IL-1beta) are shown physiological insomnia roles (6). IL-1 concentration in the cells plasma of healthy human was assumed to be the highest during awakenings and infusion and at the beginning of sleep (7). The most stable IL-1β levels in plasma with cytokine cycle variation can affect patients with narcolepsy, and interleukin-6 (IL-6) and hypersomnia (3-4). The significant increases in cytokine concentrations can be measured by OSAS and narcolepsy, and average sleep having correlation with the intensity of sleep, sleep can be measured as latency. It is associated with IL-6 levels and body mass index (BMI) (8-10). Obesity is one of the serious consequences leading to uneven course of sleep. This interaction can lead to increases in inflammatory cytokines (9). IL- 1β and IL-6 genes can alter the levels of protein synthesis. IL-6 functions are known as lipid metabolism and energy expenditure (11). Polymorphism in the promoter region of the IL-6 gene influences the level of 174 (G174C) interleukin 6. Also in the arrangement of body mass interleukin - 1β is known to be effective (12). The gold standard method in the diagnosis of sleep disordered breathing is the polysomnography method (10). Currently, the connection of OSAS syndromes with genetics is investigated (12). In our study, the genomic aspects of IL-1β and IL-6 gene polymorphisms from cytokine genes in patients with obstructive sleep syndrome were examined. The most common form of polymorphism is the single nucleotide polymorphism consisting of a single base pair change in genomic DNA. Allelic and genotypic distribution analysis of cytokine genes were evaluated in individuals with obesity and individuals with sleep apnea syndromes visiting outpatient clinics. The aim of the study was to evaluate the clinical importance of cytokine genes which have been found to be linked with OSAS. In this sense, genotypic studies polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) method was used. PCR products were sequenced with an Applied Biosystems Automated Sequencer. PATIENTS AND METHODS Patients and controls During three years ( ) 1000 people having problems of sleepiness disease reported to the sleep laboratory in Kafkas University, Turkey. Patients diagnosed with OSAS were selected for the research by the results of polysomnography (PSG), and were studied at the sleep laboratory at Kafkas University Medicine Faculty Research and Application Hospital (apnoea hypopnoea index, AHA of < 5-30). A control group of 80 voluntarily subjects with an 217

116 Medicinski Glasnik, Volume 12, Number 2, August 2015 apnoea hypopnoea index (AHI) <5 were selected randomly (general population without any classification). The population in this region shows trends toward obesity resulting from poor nutrition habits and the long winter season. In addition, sleep disease diagnoses in the region exceed the country s average (p<0.05). For the patients and controls an ethical approval was received from the Medical Ethics Committee of Ataturk University (Erzurum). Polysomnography Full-night polysomnographic recording was performed using an Embla N7000 system (Medcare; Reykjavik, Iceland). The following parameters were recorded: electroencephalography, electrocardiography, electrooculography, submental and anterior tibialis muscle electromyography, nasal pressure, oronasal airflow by thermal sensor, snoring, oxygen saturation by finger oxymeter, and respiratory effort by thoracic and abdominal inductance plethysmography (13). Sleep disordered breathing events were scored manually by the same investigator, according to the American Academy of Sleep Medicine criteria (14). Obstructive apnea was defined as a drop in the peak oronasal thermal sensor excursion by 90% from baseline for at least 10 s. Hypopnea was defined as at least a 50% drop in airflow for at least 10 s despite respiratory efforts and at least a 3% decrease in oxyhemoglobin saturation. Patients were diagnosed with OSAS if the AHI was 5. Grading was conducted according to mild OSAS with an AHI of 5-14, moderate OSAS with an AHI of 15-29, and severe OSAS with an AHI 30 (13-14). The lowest O saturation value was 2 measured throughout the night for each patient. Polysomnographic results of the patients and controls were recorded in the hospital sleep laboratory using the 24-hour duration as a baseline with the laboratory PSG system. The heights of the patients and controls were measured in cm, while weights and body mass index (BMI) values were calculated in kg/m 2. Determination of genotypes the Interleukin- 6 and Interleukin-1 For genotyping 10 ml blood samples were taken from each patient and the control group into tubes with EDTA. DNA was extracted using commercially available Invitrogen genomic DNA extraction mini kits (CS11010, London, UK) DNA purification kit according to the manufacturer s instructions. The isolated DNA samples were measured at the Nanodrop Spectrophotometer (Thermo ND1000 Wilmington, USA) and kept at -20 C. The primers used to identify Interleukin- 6 (IL-6) were F: 5 -TGA CTT CAG CTT TAC TCT TTG T-3 and R: 5 -CTC AGG TGT CCT CGAAGAAAT CAAA-3 the Interleukin-1β genes (IL-1β) specific primers were: F 5 - GCT TTT TTG CTG TGA GTC CCG-3 and R 5 -CTC AGG TGT CCT CGAAGAAAT CAAA-3 (15). A final volume 25 μl PCR protocol that included 2.5 μl 10X Taq polymerase buffer solution, 2,5μL magnesium chloride (2 mm), 2μL dntp mix (0.2 mm), 1 μl forward primer (10 pmol), 1 μl reverse primer (10 pmol), 2μL genomic DNA (100ng/μL), 1 μl DNA taq polymerase enzyme (5u/μL), and 13 μl distilled water; a total volume of 25 μl was used, PCR conditions were as follows: an initial denaturation for 5 min at 94 C, then 35 cycles at 94 C for 45 s, at 63 C for 45 s, at 72 C for 55 s, and a final extension at 1 cycle 72 C for 7 min. The PCR products were detected by agarose gel electrophoresis (at 90V, 300 A for 1.5 h) on 2% agarose gel containing ethidium bromide, and the fluorescent intensity of each band was evaluated with a UV transilluminator (Gel Logic Pro 2200, Montreal,Canada) (16) For the Interleukin-1β polymorphism, the PCR amplification bands were observed as 195 bp, and the Interleukin- 6 amplification bands as 198bp. Amplified products were digested: Interleukin- 1β with 5U Thermus aquaticus (TaqI), and Interleukin- 6 was digested with 5U Listeria welshimeri (LweI) (New England Biolabs, INC UK) (17). Digestion products were visualized, and resulting fragments were separated on 2.5% agarose gel and with ethidium bromide staining under ultraviolet illumination (Gel Logic Pro 2200, Canada). The single amplicon of 195 bp as a result of the section of the Interleukin- 1β polymorphism with the restriction enzyme (TaqI) was separated into two DNA fragments as 110 bp and 85 bp. As a result of the section of Interleukin- 6 with enzyme (LweI), the 198 bp 218

117 Gok et al. Gene polymorphisms and sleep apnoea syndrome bands were observed as having 110 bp + 88bp. The PCR products were then isolated using agarose gel electrophoresis (15-18). All of the genomic analyses were conducted in the laboratory of molecular genetics in Department of Bioengineering, Kafkas University. In addition, Bigdye Cycle Sequencing kit v.3.1, Applied Biosystems and approximately 5 μl true of PCR products were sequenced with an Applied Biosystems Automated Sequencer (ABI 3130 XL Genetic Analyzer, Foster City, CA USA). Restrictions mapping and SNP bioinformatic analysis was done as Vector NTI Software (Life Technologies). Statistical analysis For each polymorphism, deviation of the genotype frequencies in the controls from those expected under Hardy-Weinberg equilibrium was assessed using the standard χ2 test. Genotype frequencies in cases and controls were compared by χ2 tests. The genotypic-specific risks were estimated as odds ratios (ORs) with associated 95% intervals (CIs) by unconditional logistic regression. p<0.05 was considered to be significant. RESULTS The patients diagnosed with the OSAS disease from Kafkas University, School of Medicine Neurology Clinic, between August July 2014, were examined according to polysomnography data (Table 1). Table 1. Demographic characteristic of obstructive sleep apnea syndrome (OSAS) patients and control group No (%) of patients OSAS patients (AHI 5-30) (n = 62) Control group (AHI <5) (n = 80) Age Females Males SD Age Females Males SD (9.6) 5 (16.1) (54) 30 (70) (32.) 10 (32.6) (46) 13 (30) 2.83 >61 18 (59.) 16 (58.1) 1.41 > Total 31 (50) 31 (50) 0.00 Total 37 (46) 43 (54) 4.24 The percentage of OSAS patients having BMI more than 30 kg/m 2 was 66% (41 out of 62), 23 (74.1%) females and 18 (58%) males. In the control group, 42.5% (34 out of 80) of examinees had BMI less than 25 kg/m 2. Patients with OSAS mostly had AHI >30, 32 (51.6%), of which 14 (45.16%) were females and 18 (58.07%) males (Table 2). Table 2. Distribution of patients with obstructive sleep apnea syndrome (OSAS) and control group according to body mass index (BMI) and apnoea hypopnoea index (AHI) OSAS patients (BMI) (N = 62) No (%) of patients Control group (BMI ) (N = 80) BMI Range Females Males Range Females Males p (25.8) 13 (41.9) > (35.2) 21 (48.8) >30 23 (74.1) 18 (58.0) >30 24 (64.8) 22 (51.2) Total 31 (50) 31 (50) Total 37 (46.3) 43 (53.7) AHI Range Females Males Range Females Males p 5-14 (mild) 7 (22.58) 0 > 5 37 (100) 43 (100) (medium) 10 (32.25) 13 (41.93) > > (45.16) 18 (58.07) >30 0 (heavy) Total 31 (50) 31 (50) Total 37 (46.3) 43 (53.7) There were some similarities between males and females in terms of frequency of gene polymorphism. An increase of BMI resulted in more IL-1β gene polymorphism change. Genotype of IL-1β gene polymorphism showed CC genotype in nine (14.5%), CT in 26 (41.9%), TT in 27(43.5%) in OSAS patients, and CC in eight (10%), CT in 16 (20%) and TT genotype in 56 (70%) controls. In patients IL-1β polymorphic change rate was observed in 26 of 62 patients (41.9%), and the same polymorphic change rate was observed in 16 of 80 (25.8%) controls. The prevalence of polymorphic changes in IL-6 gene was 11.3% (seven of the 62) patients, and 20% (16 of 80) controls (p<0.05 for IL-1B C / T and IL-6 G174C polymorphic regions of cytokines) (Table 3). Table 3. Genotypic and allelic results of IL-1β and IL-6 gene polymorphisms in the obstructive sleep apnea syndrome (OSAS) patients and controls No (%) of patients Interleukin-1β OSAS (n=62) Control Group (n=80) Genotypes CC TT CT p CC TT CT p Frequencies of genotypes (14.5) (43.5) (41.9) (10) (70) (20) Alleles C T C T Frequencies of alleles (0.3) (0.6) (0.2) (0.8) Interleukin-6 Genotypes GG CC GC p GG CC GC p Frequencies of genotypes (77.4) (11,3) (11,3) (70) (10) (20) Alleles G C G C Frequencies of Alleles (82.3) (17.7) (80) (20) 219

118 Medicinski Glasnik, Volume 12, Number 2, August 2015 Figure 1. Sequencing IL-1β gene; A) the restriction enzyme cleavage sites are provided in control individuals (TaqI); B restriction enzyme cleavage site is provided in obstructive sleep apnea syndrome (OSAS) patients as sequencing results (MvaI) (Gok I, 2014) The most important result of DNA sequence of IL-1beta gene proved restriction enzymes (TaqI) cleavage sites for both patients and control group (Figure 1). The most important result of DNA sequence of IL-6 gene was shown as base pair s changes from GG in the control group to GC and CC in the OSAS patients, in other words SNP. Furthermore, IL-6 gene restriction enzymes (Lwe I) cleavage sites were proven for both patients and control group after sequence resulting. In the SNP regions and restriction enzyme (Lwe I) cleavage site were detected and are given in Figure 2. Figure 2. In the IL-6 gene sequencing results: A) SNP region is shown normal (T/T) in control individuals; B) and C) sequencing result of obstructive sleep apnea syndrome (OSAS) patients (SNP) region the change (G/T) is provided (Gok I, 2014) 220

119 Gok et al. Gene polymorphisms and sleep apnoea syndrome DISCUSSION In various countries around the world, OSAS is different in terms of clinical signs and the prevalence of incidence is increasing between years. Generally, the disease in populations was found to be 4% in males and 2% in females (5,19). There is sleep apnea syndrome in approximately % of Turkey s population (20-21). Yang et al. in 2013 in China gave researches importance of its genetic aspects; until now many genes and polymorphism region related to the disease have been identified (22). The diversity of genes which are responsible for the formation of respiratory-related diseases and of polymorphic region located in this gene was expected to be made a criterion for the diagnosis of OSAS and genomic studies were carried out. In addition, an increase of BMI results in a change in cytokine gene polymorphisms (17,23). In this study, according to polysomnography results in the population of Northern Anatolia, IL-1β, IL-6 polymorphism genotypes distribution was investigated in individuals diagnosed with OSAS: 87% of patients with OSAS included in the study were over 40 years of age, and 66% of the cases with BMI >30 were aged above 30 years, and our results support this relationship. In the study by Popko et al. in 2008 in Poland it was found that IL-1β polymorphism was a risk factor for obesity, IL-6 polymorphism was associated with interleukin receptor function in carrying the C allele, but there was no significant relationship with obesity in a group of 140 women, 50% of which had BMI <30 kg/m 2 (others had MBI >25 kg/m 2 (15). Our research is not in compliance with the research of Popko et al. regarding IL-6 polymorphism, however it correlates with IL-1β polymorphism. Riha et al. in 2009 in Germany and the UK, investigated IL-1 polymorphism in normal, overweight and obese man and women, and they found that polymorphisms of IL-1β can be effective on the development of obesity in European society (8). In other study, Arnardottir et al. in 2012 in Iceland, confirmed that the BMI increase resulted in IL-6 gene polymorphism change, and obesity and other respiratory disease triggers of the OSAS (23). In this study we observed an increase in body mass index of those bearing the genotypes of IL-6 and alleles of IL-1β; BMI values of 41 of 62 individuals (66%) with OSAS whose two polymorphisms in the IL-6 and IL-1β gene were examined in our study were observed of greater than 30. Our findings are similar to the results of the Mannarino et al. (14). In a study conducted by Buck and colleagues in 2010 for the IL-6 gene in Northern Europe, authors have found no relationship between IL-6 genotype and obesity (12). In this research that was carried out for North East Anatolia region, the IL-6 gene was not associated with obesity, which is in line with the findings of Buck et al. In research conducted in various European countries in a similar manner, the effect of IL-6 gene was found to be associated with metabolism and energy consumption (24-25). In our study, we suggested that IL-6 gene polymorphism had no direct relationship with OSAS, but if the BMI is greater than 30 in patients with OSAS and controls, it may increase the risk of obesity. A changing of the balance of IL-1β gene polymorphism could induce metabolism, and environmental interactions of genes may trigger the formation of OSAS (15). There is a number of genetic variants of this gene polymorphism affecting the OSAS progress of obesity or vice versa, as a cause there are or there will be probably susceptibility variants affecting the development of obesity of OSAS but also affecting pleiotropism. Identification of such genes and understanding their function will contribute to new perspectives in the partners pathogenesis of these diseases. Finally, saying that genetic polymorphisms may influence susceptibility to each disease may not be very accurate; the possibility should not be ignored. Similarly, the use of such a model with information about the genetic aspects of obesity will be able to allow for a more comprehensive way to understand how OSAS can affect the obesity. FUNDING We are grateful to the Kafkas University Scientific Research Project Unit (Grant No: FEF: Kars, Turkey) for financial support of this study. TRANSPARENCY DECLARATION Competing interests: None to declare. 221

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